Breast Cancer Cell Line Bcap-37 Research Articles

High-dose vitamin C suppresses the invasion and metastasis of breast cancer cells via inhibiting epithelial-mesenchymal transition.

PurposeVitamin C (VC) is a kind of essential nutrient in the body regarded as a canonical antioxidant during the past hundred years. However, the anti-cancer effect of VC is controversial. Our study is trying to clarify the relationship between VC dosage and breast cancer metastasis.MethodsHuman breast cancer cell lines Bcap37 and MDA-MB-453 were treated with VC at three different concentrations (low-dose, 0.01 mM; medium-dose, 0.1 mM; high-dose, 2 mM). Wound healing assays were conducted for migration assay; transwell tests were performed to detect the ability of cell invasion. The protein levels were evaluated by Western blot analysis or immunohistochemistry. Tumor xenografts in nude mice were built to test the effects of VC on breast cancer cell proliferation and metastasis.Results0.01 and 0.1 mM VC promoted cell migration and invasion when compared with the control group, but 2 mM VC significantly suppressed cell migration and invasion of breast cancer cell lines. High-dose VC increased E-cadherin and reduced Vimentin, indicating that high-dose VC suppressed epithelial-mesenchymal transition (EMT) in breast cancer cells. Besides, high-dose VC inhibited cell invasion promoted by TGF-β1 in breast cancer cells. Meanwhile, high-dose VC reversed the suppression of E-cadherin and enhancement of Vimentin induced by TGF-β1 in breast cancer cells. Furthermore, high-dose VC significantly inhibited breast cancer metastasis in vivo.ConclusionHigh-dose VC inhibits cell migration and invasion of breast cancer cell lines through suppressing EMT. Thus, it may be considered as an anticancer drug candidate for breast cancer patients.

Distinct characterization of two vinorelbine-resistant breast cancer cell lines developed by different strategies.

Resistance to chemotherapy is a major obstacle to the successful treatment of breast cancer patients. Recently, we successfully established two vinorelbine-resistant sublines, BC-DS and BC-TS, from the human breast cancer cell line BCap37, with different 'two-stage screening methods'. Interestingly, though BC-DS and BC-TS were developed from the same BCap37 cell line with the same drug, they showed remarkable differences. Compared with the parental BCap37 cells both BC-DS and BC-TS had resistance to vinorelbine, but the resistant characterizations are both unstable. BC-DS showed increased migration capability while BC-TS showed reduced migration capability. When investigating their multidrug resistance, we found BC-DS became more sensitive to methotrexate, which suggested that combination of MTX and vinorelbine could be a new treatment strategy. Moreover, BC-DS and BC-TS overexpressed P-glycoprotein at different levels. Our research also showed that the present clinical usage of vinorelbine is reasonable. These findings suggest that the vinorelbine-induced multiple drug resistance (MDR) sublines may be used as an in vitro model not only to further elucidate possible mechanisms of MDR involved in the human breast cancer, but also to find methods to optimize the curative effect of vinorelbine in clinic.

Abstract 624: Preliminary study of the follicle-stimulating hormone α/β and the pathogenesis of postmenopausal breast cancer

Abstract Objective To study the difference of serum FSH level between breast cancer and healthy people in postmenopausal women; To investigate the internal relations of FSH and the tumor clinical classfication ,lymph node metastasis, and the status of ER,PR,Her-2 and Ki67; To study the expression of FSHR in breast cancer cell line and breast cancer tissue ; To study whether rhFSH induce proliferation and metastasis of breast cancer cell line and the exact mechanism in vitro and in vivo. Method Part 1: Clinical datas of 187 postmenopausal women diagnosed of breast cancer and 374 postmenopausal healthy people were collected for statistic analysis. Part2: Two breast cancer cell line (Bcap-37 and MDA-MB-231) have been used to study whether the rhFSH induce proliferation and metastasis in vitro. Part3: In vivo study of the rhFSH whether to promote tumor proliferation.We established a postmenopausal animal model of Balb/c nu/nu mouse by ovariectomized (OVX). The experiment was divided into four groups: Sham , OVX control, using GnRha after OVX, using GnRHa followed rhFSH after OVX. Part 4: RT-PCR and Reverse-PCR has used to investigate the expression of FSHR in many breast cancer cell line. The expression of FSHR in 17 samples of breast tissue was also been detected by RT-PCR. Results Part1: The serum level of FSH ,LH in postmenopausal breast cancer were higher than postmenopausal healthy people( 61.83±1.475 vs. 48.13±1.256 IU/L;27.83±0.74 vs.23.88±0.6499 IU/L).FSH has a positive correlation with Ki67 and Her-2 expression in breast . Part2: It has been found that rhFSH could promote the Bcap-37 and MDA-MB-231cell proliferation regardless of whether low concentration (10 ng/mL) or high concentration (50 ng/mL) in vitro. The rhFSH induce the proliferation and metastasis by activated ERFR/ERK1/2 cell pathway. Part3: A high concentration of rhFSH (≈100IU/L) could promote breast cancer cell line Bcap-37 and MDA-MB-231 proliferation in vivo. Part4: The FSHR was not expressed in all of the breast cancer cells tested and breast cancer tissue. Conclusion Postmenupausal breast cancer patient favored a high serum gonadotropin level compared with healthy people.FSH has a positive correlation with the Her-2 and Ki 67expresssion in breast cancer. The rhFSH cound promote the proliferation and metastasis of breast cancer cell line Bcap-37 and MDA-MB-231 by activated the EGFR/ERK1/2 pathway. A high concentration of rhFSH (≈100IU/L) may also promote the proliferation of breast cancer cell line in vivo. However, there is no FSHR expression in breast cancer cell line and breast cancer tissue. Further studies are needed to charlify the exact mechanism between FSH and postmenopausal breast cancer. Note: This abstract was not presented at the meeting. Citation Format: Yi-Ding Chen. Preliminary study of the follicle-stimulating hormone α/β and the pathogenesis of postmenopausal breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 624. doi:10.1158/1538-7445.AM2014-624

Anticancer mechanisms of temporin-1CEa, an amphipathic α-helical antimicrobial peptide, in Bcap-37 human breast cancer cells

AimsTemporin-1CEa, a 17-residue antimicrobial peptide, is known to exert broad-spectrum anticancer activity that acts preferentially on cancer cells instead of normal cells. However, the mechanism of cancer cell death induced by temporin-1CEa is weakly understood. Main methodsHere, we investigated the cytotoxic and membrane-disrupting effects of temporin-1CEa on human breast cancer cell line Bcap-37, using MTT assay, electronic microscope observation, fluorescence imaging and flow cytometry analysis. Key findingsThe MTT assay indicated that one-hour temporin-1CEa treatment led to rapid cell death in either caspase-dependent or -independent manner. The electronic microscope observation suggested that temporin-1CEa exposure resulted in profound morphological changes in Bcap-37 cells. The fluorescence imaging and flow cytometry analysis demonstrated that temporin-1CEa exhibited membrane-disrupting property characterized by induction of cell-surface phosphatidylserine exposure, elevation of plasma membrane permeability, and rapid transmembrane potential depolarization. Moreover, temporin-1CEa might also induce rapid cell death through mitochondria-involved mechanisms, including rapid intracellular Ca2+ leakage, collapse of mitochondrial membrane potential (Δφm) and over-generation of reactive oxygen species (ROS). SignificanceIn summary, the present study indicates that temporin-1CEa triggers a rapid cytotoxicity in Bcap-37 cells through membrane-destruction and intracellular mechanisms involving mitochondria. These intracellular mechanisms and direct membrane-destruction effect were evaluated helping to understand the detail action of antimicrobial peptides in mammalian cancer cells.

Different administration strategies with paclitaxel induce distinct phenotypes of multidrug resistance in breast cancer cells

Both dose-dense and dose-escalation chemotherapy are administered in clinic. By approximately imitating the schedules of dose-dense and dose-escalation administration with paclitaxel, two novel multidrug resistant (MDR) cell lines Bads-200 and Bats-72 were successfully developed from drug-sensitive breast cancer cell line BCap37, respectively. Different from Bads-200, Bats-72 exhibited stable MDR and significantly enhanced migratory and invasive properties, indicating that they represented two different MDR phenotypes. Our results showed that distinct phenotypes of MDR could be induced by altered administration strategies with a same drug. Administrating paclitaxel in conventional dose-escalation schedule might induce recrudescent tumor cells with stable MDR and increased metastatic capacity.

Antiproliferative and cell apoptosis-inducing activities of compounds from Buddleja davidii in Mgc-803 cells

BackgroundBuddleja davidii is widely distributed in the southwestern region of China. We have undertaken a systematic analysis of B. davidii as a Chinese traditional medicine with anticancer activity by isolating natural products for their activity against the human gastric cancer cell line Mgc-803 and the human breast cancer cell line Bcap-37.ResultsTen compounds were extracted and isolated from B. davidii, among which colchicine was identified in B. davidii for the first time. The inhibitory activities of these compounds were investigated in Mgc-803, Bcap-37 cells in vitro by MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, and the results showed that luteolin and colchicine had potent inhibitory activities against the growth of Mgc-803 cells. Subsequent fluorescence staining and flow cytometry analysis indicated that these two compounds could induce apoptosis in Mgc-803 cells. The results also showed that the percentages of early apoptotic cells (Annexin V+/PI-, where PI is propidium iodide) and late apoptotic cells (Annexin V+/PI+) increased in a dose- and time-dependent manner. After 36 h of incubation with luteolin at 20 μM, the percentages of cells were approximately 15.4% in early apoptosis and 43.7% in late apoptosis; after 36 h of incubation with colchicine at 20 μM, the corresponding values were 7.7% and 35.2%, respectively.ConclusionsColchicine and luteolin from B. davidii have potential applications as adjuvant therapies for treating human carcinoma cells. These compounds could also induce apoptosis in tumor cells.

Effect of Norcantharidin on the Human Breast Cancer Bcap-37 Cells

Norcantharidin (NCTD), a chemically modified form of cantharidin, is a potential anticancer drug. In this study, the effects of NCTD on the cellular viability, reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and DNA damage in the human breast cancer cell line Bcap-37 were investigated with confocal and fluorescence microscopy. The cell cycle was further analyzed using the CellQuest software of a Becton-Dickinson FACS flow cytometer. The results indicated that the cellular viability was decreased with the growing concentrations of NCTD and time exposure. Moreover, the fluorescence intensity of ROS was increased, whereas the MMP was decreased in Bcap-37 cells with the growing concentrations of NCTD. NCTD induced a dose-dependent DNA damage and reduced the G1 peak in Bcap-37 cells. The G2/M peak of Bcap-37 was also decreased by the higher concentration of NCTD.

Influence of TROP-2 gene on the ability of human breast cancer cell metastasis

Objective To study the effects of tumor-associated calcium signal transducer-2 (TROP-2) gene small interfering RNA(siRNA) on adhesion and invasion of human breast cancer cell. Methods Real time PCR was used to evaluate the TROP-2 mRNA of seven human breast cancer cell lines Bcap-37 ,LCC1 ,MCF-7 ,MDA-MB-231,MDA-MB-435, MDA-MB-468 ,and ZR75-1. The cell line of TROP-2 highest expression was transfected with different dose of TROP-2 siRNA. The expression of TROP-2 mRNA and protein were determined by Real-time quantitative PCR and immumoflurescence method. The cell adhesion was evaluated by MTT assay,and invasion was exmined by hoyden chamber,respectively. Results Cell line MCF-7 showed the highest elevation of TROP-2 mRNA in seven breast cancer cell lines. The results from real-time quantitative PCR and immumoflurescence method showed that TROP-2 mRNA and protein reduced in time-and dose-dependent manners( P ＜ 0.01 ;P ＜ 0.01 ). The adhesive rate of siRNA groups(5 nM,10 nM,and 20 nM)was(52.9 +2.5)％ ,(25.6 ±2.3)％, ( 12.8 +2.2)％ (P ＜0.01 ) ,respectively.The transwell results showed that the invasion cells was(78 ± 17), (39 ± 15), ( 19 ± 16), ( 136 +25 ) and( 139 ±21 )in different groups(5,10,20 nM siRNA,and controls) ,respectively(P ＜0.01). Conclusion TROP-2 gene might play an important role in adhesion and invasion of human breast cancer cell. siRNA targeted TROP-2 could effectively inhibit adhesion and invasion of human breast cancer cell. Key words: Breast Neoplasms; Tumor-associated calcium signal transducer-2

HSF1 overexpression enhances oncolytic effect of replicative adenovirus

BackgroundE1B55kD deleted oncolytic adenovirus was designed to achieve cancer-specific cytotoxicity, but showed limitations in clinical study. To find a method to increase its efficacy, we investigated the correlation between oncolytic effect of such oncolytic adenovirus Adel55 and intracellular heat shock transcription factor 1 (HSF1) activity.MethodsIn the present study, human breast cancer cell line Bcap37 was stably transfected with constitutively active HSF1 (cHSF1) or HSF1 specific siRNA (HSF1i) to establish increased or decreased HSF1 expression levels. Cytotoxicity of Adel55 was analyzed in these cell lines in vitro and in vivo. Furthermore, Adel55 incorporated with cHSF1 (Adel55-cHSF1) was used to treat various tumor xenografts.ResultsAdel55 could achieve more efficient oncolysis in cHSF1 transfected Bcap37 cells, both in vitro and in vivo. However, inhibition of HSF1 expression by HSF1i could rescue Bcap37 cell line from oncolysis by Adel55. A time course study of viral replication established a correlation between higher replication of Adel55 and cytolysis or tumor growth inhibition. Then, we constructed Adel55-cHSF1 for tumor gene therapy and demonstrated that it is more potent than Adel55 itself in oncolysis and replication in both Bcap37 and SW620 xenografts.ConclusionscHSF1 enhances the Adel55 cell-killing potential through increasing the viral replication and is a potential therapeutic implication to augment the potential of E1B55kD deleted oncolytic adenovirus by increasing its burst.

23,24-Dihydrocucurbitacin B induces G 2/M cell-cycle arrest and mitochondria-dependent apoptosis in human breast cancer cells (Bcap37)

23,24-Dihydrocucurbitacin B (DHCB), a cucurbitacin-derived compound known to posses anticancer and anti-inflammatory activities. In this study, DHCB, isolated from roots of Trichosanthes kirilowli which is a traditional Chinese herb medicine used as treatments for cancer and other diseases, has been found to inhibit the proliferation of human cancer cell lines Bcap37, HeLa, SW620, SMMC-7721, K562 and MCF-7 in a dose- and time-dependent manner, and induce apoptosis in human breast cancer cell line Bcap37 at low concentration. DHCB-induced Bcap37 apoptosis was characterized with the changes in nuclear morphology, DNA fragmentation, activation of caspase-like activities, poly(ADP-ribose) polymerase cleavage, release of cytochrome c into cytosol. The cell death was partly prevented by a caspase-family inhibitor Z-VAD-FMK. The results suggest that DHCB-induced Bcap37 apoptosis through mitochondrial dependent pathway. Flow cytometric analysis revealed that at the lower dose of 1.8 and 3.6 μM, DHCB-induced cancer cell lines death via an apoptotic process rather than necrotic one; whereas, the higher dose of 8.9, 17.9 and 35.7 μM induced cell death via the necrotic process. Cell-cycle analysis demonstrated DHCB induction of G 2/M phase cell-cycle arrest and apoptosis. The overall results suggest that DHCB might have the therapeutic value against human cancer cell lines, especially the breast cancer cell lines.

High-level expression, purification and pro-apoptosis activity of HIV-TAT-survivin (T34A) mutant to cancer cells in vitro

Survivin, a novel member of the IAP family, was observed to express in the most common human cancers. Anti-cancer therapy targeting survivin had drawn considerable attention. This study focused on high-level expression of recombinant protein TAT-survivin (T34A) mutant in E. coli, purification and bioactivity of pro-apoptosis to various cell lines in vitro. The cDNA encoding survivin was cloned by RT-PCR from breast cancer cell lines B-cap37. After PCR site-directed mutagenesis and construction of expression vector pRSET-B-TAT-survivin (T34A), targeted TAT-survivin (T34A) protein was expressed highly in E. coli BL21 (DE3) by 0.5mM IPTG induction and its yield could reach 650 mg/l in fermentation culture. The fusion protein in a form of inclusion body was then solubilized, refolded and purified to a purity of 98% by cation exchange chromatography and size-exclusion chromatography. Four hundred and eighty milligrams protein of interest was obtained in per liter fermentation culture. This showed that the efficient procedures of large-scale expression and purification were successful for the mass production of the recombinant protein. Pro-apoptosis effects of target protein on four cancer cell lines and one normal cell line from human were confirmed by the change of morphology, and pro-apoptosis activity was evaluated by MTT, fluorescent staining of nuclei and flow cytometry assay. Results indicated that B-cap37 and SW1990 were very sensitive to TAT-survivin (T34A) protein. This finding revealed the recombinant protein was promising as an anti-cancer drug.

Construction, expression, and purification of HIV–TAT-survivin (T34A) mutant: A pro-apoptosis protein in Escherichia coli

As a novel member of the IAP family, survivin was observed to express in the most common human cancers. Anti-cancer therapy targeting survivin has drawn considerable attention. This report presented firstly construction of recombinant plasmid pRSET-B-TAT-survivin (T34A), expression in Escherichia coli, purification, renaturation, and bioactivity. The cDNA encoding survivin was cloned by RT-PCR from breast cancer cell lines B-cap37. Expression vector pRSET-B-TAT-survivin (T34A) was constructed by PCR after survivin was mutated by PCR site-directed mutagenesis. Recombinant TAT-survivin (T34A) protein was expressed highly in E. coli BL21 (DE3) by 0.5 mM IPTG induction and its yield could reach 650 mg/l in fermentation culture. The fusion protein in a form of inclusion body was then solubilized, refolded, and purified to a purity of 98% by cation exchange chromatography and size-exclusion chromatography. Fourhundred and eighty milligrams protein of interest was obtained in per liter fermentation culture. The protein of interest was identified by SDS–PAGE and Western blot analysis, and great bioactivity of target protein to two cancer cell lines was confirmed by morphological changes and evaluated by MTT. The findings suggested that recombinant protein TAT-survivin (T34A) has a bright future in cancer therapy targeting towards survivin, and the efficient procedure of expression and purification may be useful for the mass production of this therapeutically important protein.

Molecular cloning and bioinformatics analysis of a novel spliced variant of survivin from human breast cancer cells

Survivin gene and its two alternatively spliced variants, survivin-2 B and survivin- Delta Ex3 gene were cloned from human breast cancer cell lines B-cap37 firstly. A new gene designated as survivin-image (SI) was cloned from above cell lines, which has not been reported yet to clone from any cell lines. It was found that the novel gene 507 bp comprises partial survivin gene (345 bp), partial image gene (155 bp) of eye cancer and other insertion of 7 bp by analyzing with a series of recent bioinformatics software at the level of nucleotide and protein deduced. Predicted 3-D structures of the new molecule showed greatly similar to that of survivin in N-terminal containing BIR by homology modeling. These results suggested SI gene (GenBank accession No.AY830084) might be a novel alternatively spliced isoforms of the survivin gene involved in other functional significances related to tumorigenesis.

HSF1 blockade-induced tumor thermotolerance abolishment is mediated by JNK-dependent caspase-3 activation

We previously blocked the heat shock transcription factor 1 function with a dominant-negative mutant (mHSF1) in breast cancer cell line Bcap37, and found that mHSF1 sensitizes Bcap37 cells to hyperthermia by promoting the apoptotic process. Here we studied the mechanism of this abolishing process and how thermotolerance develops in Bcap37 cells. The results indicated that mHSF1 abolished acquired or intrinsic thermotolerance in Bcap37 cells by enhancing JNK and caspase-3 pathways, two stress-induced apoptotic pathways, after hyperthermia, and interference with either one of them attenuated hyperthermia-induced apoptosis. Furthermore, epistasis assay of these two pathways suggested that JNK was upstream of the caspase-3 pathway. Conversely, other hyperthermia-induced kinases implicated in cell survival and death, Akt, ERK or p38, did not influence the effect of mHSF1, indicating that these kinases were not implicated in this abolishing process. In addition, we found that the development of acquired thermotolerance of Bcap37 cells was associated with the suppression of JNK activation after mild preheat treatment and was not reduced by Akt, ERK or p38 inhibition. In contrast, the intrinsic thermotolerance of Bcap37 cells was due to the intrinsic high levels of Akt and ERK activities since Akt or ERK inhibition resulted in increased thermosensitivity of Bcap37 cells. Our results suggest that mHSF1 plays a valuable role in the thermotolerance abolishment of Bcap37 cells, which likely contributes to tumor therapy in combination with hyperthermia.

Study on interleukin-18 gene transfer into human breast cancer cells to prevent tumorigenicity.

To study the effect of interleukin-18 gene transfection on the tumorigenesis of breast cancer cell line Bacp37, human breast cancer cell line Bcap37 were transfected with Lipofectamine and selected by G418. The biological expression of rhIL-18 was tested by RT-PCR and ELISA method; nude mice were injected with Bcap37 cell with or without the hIL-18 gene. The hIL-18 cDNA was successfully integrated into Bcap37 cell; 126.3+/-4.5 pg hIL-18 secreted by one million transduced cells in 24 hours. Nude mice injected with IL-18 gene engineered Bcap37 cell had no tumor growth. These findings indicated that human breast cancer cells were successfully modified by the gene of IL-18 cytokine; the IL-18 gene engineered Bcap37 cells secreted hIL-18 and lost their tumorigenicity. The Bcap37 cells transduced with IL-18 gene may be used as breast cancer vaccine.

Establishment of a P-glycoprotein substrate screening model and its preliminary application.

To establish a high P-glycoprotein (P-gp) expressing cell line as a model for studying drug absorption and distribution, and to explore the preliminary application of this screening model. A full-length MDR1 cDNA fragment in plasmid pMDRA1 was first subcloned into plasmid pET28a(+), then MDR1 cDNA was cut from the recombinant plasmid with double-digestion and ligated into the mammalian expression vector pcDNA3.1(+). The recombinant plasmid pcDNA3.1(+)/MDR1 was transfected into breast cancer cell line Bcap37 using the Superfect transfection reagent. Several stably transfected clones were obtained after selection with G418. Real-time fluorescent quantitative RT- PCR and Western blot methods were used to detect the expression of P-gp, and the cellular location of the expressed protein was determined by immunohistochemical staining. Drug sensitivity assay was used to evaluate the biological function of expressed P-gp. Concentration of quercetin in cells was determined by high-performance liquid chromatography (HPLC). The recombinant plasmid was confirmed to be inserted in the correct orientation by restrictive enzyme digestion and DNA sequencing. Real-time fluorescent quantitative RT-PCR showed a higher level of P-gp mRNA in transfected cells compared to that in the control cells, and the Western blot result also indicated that P-gp expression in transfected cells was higher than that in control cells. The immunohistochemical staining showed that the expressed P-gp was localized on cell membranes. Drug sensitivity assay showed that the IC50 for adriamycin and colchicine of the transfected cells was higher than that of the control cells. The concentration of quercetin in model cells was lower than that in control cells by HPLC. After P-gp inhibitor verapamil was administered, the concentration of quercetin in model cells was increased. A high P-gp expressing cell line can be established, which could provide a suitable in vitro model system for studying drug intestinal absorption mechanism, predicting the drug permeability characteristics and screening new multi-drug resistance reversing agents. With this model, quercetin can be found to be transported by P-gp, and it is a P-gp substrate.