A Fluoro Derivative of Embelin, as Potent B-RAF Inhibitor in Melanoma

Background: Melanoma is one of the common forms of skin cancer and B-RAF is a mutated protein found in most Melanomas. The important function of B-RAF is normal cell growth and survival. Most of known B-RAF mutations are V600E mutations. Vemurafenib is the currently used fluorine based drug used for V600E mutations but this drug has side effects, so in this scenario, more potent drugs with less side effects are required. Objective: This study aims to develop a more effective lead compound as B-RAF inhibitor from a hydroxyquinone, by structural modification of embelin, a naturally occurring hydroxybenzoquinone, which has got a potency of detoxifying blood, hence useful in wide range of skin diseases. Thus a fluorine substituted semisynthetic derivative of embelin, 5-(3-chloro-4-trifluoromethoxy phenyl amino)-2- hydroxy-3-undecyl- [1, 4] benzoquinone to fight against skin cancer was prepared. Method: Fluoro derivative of embelin was synthesized by the direct condensation of embelin with 3-chloro-4-trifluoromethoxy aniline. The structure of the product was characterized using various spectral data, obtained from IR, 1H NMR, 19F NMR, 13C NMR and Mass spectrum. Various in vitro studies like Antiproliferative study in A375 Cell Lines, (B-RAF Elisa), Western Blotting analysis, Gene Expression study by Reverse Transcriptase PCR, Caspase assay, Flow Cytometry analysis, Clonogenic assay and Transwell Migration assay were carried out to find its biological activity. Results: A semisynthetic derivative of embelin 5-(3-chloro-4-trifluoromethoxy phenyl amino)-2-hydroxy-3-undecyl- [1, 4] benzoquinone (EOCF) was prepared and the structure of the derivative was confirmed by spectral analysis. The MTT assay proves that the fluoro derivative of embelin exhibited better anticancer activity in Melanoma cell lines than the parent compound, embelin. Western blot analysis showed that B-RAF expression level was reduced by the addition of derivative than the parent compound embelin. The Caspase ELISA analysis indicated that the derivative was found to be a good apoptotic marker and from the flow cytometry analysis, it was observed that the cell arrest occurs at G0/G1 phase. Its antimetastatic activity determined using Clonogenic assay indicated that the derivative, EOCF inhibits the metastatic effects in melanoma cell lines. Migratory potential of Melanoma cells were significantly reduced in presence of EOCF when Transwell migration assay was conducted. Conclusion: This work established that the potency of the synthesized compound was more than the parent compound, embelin when it was structurally modified with 3-chloro-4-trifluoromethoxy aniline and that the derivative can be used as a lead molecule for further drug discovery.

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An augmentation of Fas (CD95/APO-1) antigen induced by radiation: flow cytometry analysis of lymphoma and leukemia cell lines.

International Journal of Molecular Medicine ◽

10.3892/ijmm.3.3.275 ◽

1999 ◽

Cited By ~ 2

Author(s):

A Nishioka ◽

Y Ogawa ◽

I Kubonishi ◽

S Kataoka ◽

N Hamada ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Lines ◽

Leukemia Cell ◽

Flow Cytometry Analysis ◽

Leukemia Cell Lines

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Phospholipase A2 from krait Bungarus fasciatus venom induces human cancer cell death in vitro

PeerJ ◽

10.7717/peerj.8055 ◽

2019 ◽

Vol 7 ◽

pp. e8055 ◽

Cited By ~ 3

Author(s):

Thien V. Tran ◽

Andrei E. Siniavin ◽

Anh N. Hoang ◽

My T.T. Le ◽

Chuong D. Pham ◽

...

Keyword(s):

Flow Cytometry ◽

Cell Death ◽

Cancer Cells ◽

Cell Lines ◽

Phospholipase A ◽

Human Lung Cancer ◽

Flow Cytometry Analysis ◽

Mcf7 Cells ◽

Ki 67 ◽

Cellular Marker

Background Snake venoms are the complex mixtures of different compounds manifesting a wide array of biological activities. The venoms of kraits (genus Bungarus, family Elapidae) induce mainly neurological symptoms; however, these venoms show a cytotoxicity against cancer cells as well. This study was conducted to identify in Bungarus fasciatus venom an active compound(s) exerting cytotoxic effects toward MCF7 human breast cancer cells and A549 human lung cancer cells. Methods The crude venom of B. fasciatus was separated by gel-filtration on Superdex HR 75 column and reversed phase HPLC on C18 column. The fractions obtained were screened for cytotoxic effect against MCF7, A549, and HK2 cell lines using colorimetric assay with the tetrazolium dye MTT- 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The primary structure of active protein was established by ultra high resolution LC-MS/MS. The molecular mechanism of the isolated protein action on MCF7 cells was elucidated by flow cytometry. Results MTT cell viability assays of cancer cells incubated with fractions isolated from B. fasciatus venom revealed a protein with molecular mass of about 13 kDa possessing significant cytotoxicity. This protein manifested the dose and time dependent cytotoxicity for MCF7 and A549 cell lines while showed no toxic effect on human normal kidney HK2 cells. In MCF7, flow cytometry analysis revealed a decrease in the proportion of Ki-67 positive cells. As Ki-67 protein is a cellular marker for proliferation, its decline indicates the reduction in the proliferation of MCF7 cells treated with the protein. Flow cytometry analysis of MCF7 cells stained with propidium iodide and Annexin V conjugated with allophycocyanin showed that a probable mechanism of cell death is apoptosis. Mass spectrometric studies showed that the cytotoxic protein was phospholipase A2. The amino acid sequence of this enzyme earlier was deduced from cloned cDNA, and in this work it was isolated from the venom as a protein for the first time. It is also the first krait phospholipase A2 manifesting the cytotoxicity for cancer cells.

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Characterization of four melanoma cell lines with electron microscopy, immunocytochemistry, cytogenetics, flow cytometry, and southern analysis

Cancer Genetics and Cytogenetics ◽

10.1016/0165-4608(92)90248-7 ◽

1992 ◽

Vol 62 (2) ◽

pp. 111-123 ◽

Cited By ~ 6

Author(s):

I. Köpf ◽

U. Stierner ◽

Q. Islam ◽

U. Delle ◽

L-G. Kindblom ◽

...

Keyword(s):

Electron Microscopy ◽

Flow Cytometry ◽

Cell Lines ◽

Melanoma Cell ◽

Southern Analysis ◽

Melanoma Cell Lines

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Methylation regulation of MUC6 correlates with metastasis of gastric cancer

10.21203/rs.2.23506/v1 ◽

2020 ◽

Author(s):

Ding Shi ◽

Xiaoxia Xi

Keyword(s):

Cell Lines ◽

Promoter Region ◽

Quantitative Polymerase Chain Reaction ◽

Migration And Invasion ◽

Sgc7901 Cell ◽

Transwell Migration Assay ◽

Migration Assay ◽

Normal Tissues ◽

Polymerase Chain ◽

Cancer Tissues

Abstract Background: The aim of this study was to investigate the mechanism of the downregulation of MUC6 and its influence on GC metastasis.Methods: The expression of MUC6 was examined in cancer tissues and their corresponding adjacent normal tissues in 40 gastric adenocarcinoma patients. The investigation of methylation level of MUC6 promoter region in gastric cell lines and gastric specimen tissues was performed through immunohistochemistry and/or quantitative polymerase chain reaction (qPCR)s. MUC6 was knocked down in GES-1 cell lines and overexpressed in SGC7901 cell lines; the effects of MUC6 knockdown and overexpression on cell migration and invasion were examined using Transwell migration assay. The effects of demethylation and methylation on MUC6 expression were examined using Western blot, qPCR, or double luciferase report experiment.Results: The expression of MUC6 in GC tissues was significantly lower than that in normal paracancerous tissues. While the cells migration and invasion abilities were decreased significantly after overexpression of MUC6, these abilities increased significantly after the knocking down of MUC6. The methylation levels of MUC6 in GC tissues and GC cell lines (MGC803, MKN45, AGS, SGC7901, and BGC823) were significantly higher than those in paracancerous tissues and gastric epithelial cells. The promoter methylation could significantly reduce the binding of MUC6 promoter region to the related transcription factors. The expression of MUC6 increased with the concentration of demethylated drugs and the time of action.Conclusion: The expression of MUC6 was regulated by methylation of its promoter, and this methylation of MUC6 promoter may lead to significant downregulation of MUC6 in GC and promote the metastasis of GC.

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Cytotoxicity of Ocimum basilicum and Impatiens walleriana Extracts on AGS and SKOV-3 Cancer Cell Lines by Flow Cytometry Analysis

International Journal of Cancer Management ◽

10.5812/ijcm.102610 ◽

2021 ◽

Vol In Press (In Press) ◽

Author(s):

Parichehr Hanachi ◽

Fariba Rezaei Fakhrnezhad ◽

Roshanak Zarringhalami ◽

Ilkay Erdogan Orhan

Keyword(s):

Flow Cytometry ◽

Cell Line ◽

Cell Lines ◽

Cancer Cell ◽

Cytotoxic Effect ◽

Cancer Cell Line ◽

Cancer Cell Lines ◽

Ocimum Basilicum ◽

Flow Cytometry Analysis ◽

Impatiens Walleriana

Background: Numerous studies investigate finding new drug candidates with an increasing death rate caused by cancer. Nowadays, herbal medicine has been noticed again because of the many side effects of chemical drugs. Objectives: In the current study, anthocyanin and carotenoid types of compounds of Ocimum basilicum and Impatiens walleriana were determined and their cytotoxic effect on human gastric adenocarcinoma (AGS) and human ovarian carcinoma (SKOV-3) cancer cell lines were investigated. The cytotoxic effect of I. walleriana on cancer cells has not been reported so far. Methods: The amount of anthocyanin and carotenoid derivatives in these two plant species were investigated by biochemical tests, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and flow cytometry methods were applied for the cytotoxicity effect of the extracts on the AGS and SKOV3 cancer cell lines. Cell necrosis and apoptosis were determined by annexin V-fluorescent isothiocyanate/propidium iodide (FITC/PI) staining and quantification by flow cytometry. Results: O. basilicum and I. walleriana contained a noticeable amount of the mentioned compounds. According to the results, the lowest IC50 (Half- maximal inhibitory concentration) value with an amount of 2.5 ± 0.21 mg/mL that indicates the most cytotoxic extract on the AGS cancer cell line belonged to I. walleriana extract. Besides, the lowest IC50 value of O. basilicum was about 0.9 ± 0. 11 mg/mL on the SKOV3 cancer cell line. The flow cytometry analysis has also proved that the toxicity of O. basilicum is more than I. walleriana on the SKOV3 cell line and the toxicity of I. walleriana was higher than O. basilicum on the AGS cancer cell line. Conclusions: O. basilicum and I. walleriana contain antioxidant compounds, which showed the cytotoxic effect on AGS and SKOV3 cancer cell lines. Further studies on animal models and subsequent trials are necessary for revealing the full potential of the extracts.

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Regulation of the New CXCR7 Receptor in Plasma Cell Dyscrasias.

Blood ◽

10.1182/blood.v110.11.3527.3527 ◽

2007 ◽

Vol 110 (11) ◽

pp. 3527-3527

Author(s):

Anne-Sophie Moreau ◽

Xavier Leleu ◽

Xiaoying Jia ◽

Judith Runnels ◽

Costas Pitsillides ◽

...

Keyword(s):

Bone Marrow ◽

Flow Cytometry ◽

Tumor Cells ◽

Cell Lines ◽

The Other ◽

Pcr Analysis ◽

Boyden Chamber ◽

Embryonic Cells ◽

Rt Pcr ◽

Migration Assay

Abstract Background: Multiple Myeloma (MM) and Waldenstrom Macroglobulinemia (WM) are characterized by widespread involvement of the bone marrow (BM) as the result of successful homing, engraftment and growth of tumor cells at that site. The receptor for SDF-1, CXCR4 has already been implicated by us and others in the migration and homing of tumor cells to the bone marrow. Recently, a new receptor for the chemokine SDF-1 has been described, namely CXCR7. To date, it has been implicated in the migration of embryonic cells during neurogenesis in zebrafish. It has been reported to be expressed in cancer cells but its function remains unknown. We have previously shown that disruption of the CXCR4/SDF-1 axis interferes with homing of MM cells to the BM. To better understand the homing and migration of MM and WM tumor cells, we sought to study the expression and function of CXCR7. Methods: MM (U266, MM1.S, RPMI, OPM2, OPM1, KMS12BM) and WM (BCWM.1 and WSU-WM) cell lines were used. CD19+ and CD138+ cells were extracted from patient bone marrow samples by microbeads selection after informed consent. To determine the expression of CXCR7 on MM and WM cell lines as well as primary samples, we used flow cytometry and RT-PCR analysis. The migration of tumor cells towards SDF-1 was studied using the transwell boyden chamber migration assay. The adhesion of tumor cells to fibronectin using a fluorometric assay. The CXCR7 inhibitor CCX-754 (ChemoCentryx Inc., Mountain View, CA) was used. Results: CXCR7 was expressed in all cell lines tested except WSU-WM by flow cytometry and RT-PCR. Interestingly, U266 cell line did not express surface CXCR4 and expressed CXCR7 and therefore was used in further experiments to determine the differential role of CXCR7 in SDF-1 related function. Primary WM (n=14) and MM (n=7) cells expressed low intensity CXCR7 by flow cytometry. Use of the above cells in experiments utilizing small molecule antagonists of both CXCR4 and CXCR7 suggested that inhibiting the binding of SDF-1 to one receptor could impact the signaling functions of the other. Experiments are ongoing to clarify the nature of the interaction between these two SDF receptors. Furthermore, adhesion of U266 cells to fibronectin was increased in presence of SDF-1, and inhibited in the presence of CCX-7754. Further analysis to examine CXCR7 and CXCR4 antagonism in adhesion are ongoing. Conclusion: we showed for the first time CXCR7 expression on MM and WM tumor cells. Our results lead us to conclude that through binding of a shared ligand, CXCR7 and CXCR4 may modulate the biological activity of the other.

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PR1 Is Cross-Presented By Multiple Myeloma Cells in Patients and Renders Multiple Myeloma Susceptible to PR1-CTL and Anti-PR1/HLA-A2 Antibody

Blood ◽

10.1182/blood.v124.21.2133.2133 ◽

2014 ◽

Vol 124 (21) ◽

pp. 2133-2133

Author(s):

Gheath Alatrash ◽

Pariya Sukhumalchandra ◽

Mao Zhang ◽

Celine Kerros ◽

Anna Sergeeva ◽

...

Keyword(s):

Multiple Myeloma ◽

Flow Cytometry ◽

Cell Lines ◽

Conflicts Of Interest ◽

Peptide Vaccine ◽

Brefeldin A ◽

Significant Inhibition ◽

Flow Cytometry Analysis ◽

Cross Presentation ◽

Time Point

Abstract PR1 is an HLA-A2-retricted, nonameric peptide that is derived from the azurophil granule proteases neutrophil elastase (NE) and proteinase 3 (P3). PR1 has been targeted successfully in acute (AML) and chronic (CML) myeloid leukemia using anti-PR1/HLA-A2 antibody (8F4), PR1-peptide vaccine and PR1-specific cytotoxic T lymphocytes (PR1-CTL). We have previously reported that NE and P3 are cross-presented by normal B cells and dendritic cells (DC), leading to PR1 expression by HLA-A2. Since multiple myeloma (MM) is a B cell malignancy, we investigated whether MM cells can cross-present PR1 as a possible target for immunotherapy. To study whether PR1 is presented by MM cells, patient bone marrow (BM) was stained with 8F4 antibody and then imaged using confocal microscopy. PR1/HLA-A2 was detected on the surface of CD138+ MM cells in BM samples from 3 of 6 HLA-A2+ MM patients (Fig. 1). We then investigated whether cellular immunity to PR1 is detected in peripheral blood (PB) from patients with MM. PB samples from MM patients who had undergone allogeneic (allo) (n=9) and autologous (auto) (n=2) stem cell transplantation (SCT) were stained with PR1/HLA-A2 dextramer in addition to standard lineage markers. PR1-specific CD8+CTL were detected in PB of 10 of 11 patients (range=0.02%-2.9%). Because P3 and NE expression is limited to myeloid cells, we sought to determine the mechanism of PR1 presentation in MM. We performed RT-PCR and western blotting on seven MM cell lines, including U266, ARK, ARP-1, OPM-2, LP-1, IM-9 and RPMI 8226. Neither NE nor P3 were detected in the MM cell lines studied at either the mRNA or proteins levels. We then investigated whether MM cells took up NE and P3. We cultured MM cells for 30 hours with 10 ug/mL of soluble NE and P3 or irradiated HLA-A2 negative PMN, the latter as a source for cell-associated NE and P3. Cells were then stained intracellularly for NE and P3 at different time points. Flow cytometry analysis showed that all the cell lines analyzed took up NE and P3. Uptake was seen as early as 1 hour after co-culture with soluble NE and P3 and was higher for soluble P3. Additionally, more uptake was seen in the cells that were co-cultured with irradiated PMN in comparison with soluble NE and P3. We then investigated whether PR1 expression in MM was through NE and P3 cross-presentation. We focused our studies on the HLA-A2+ U266 MM cell line. U266 cells were co-cultured with soluble NE, P3 or irradiated PMN, as described in the previous section, and then surface stained with 8F4. We detected PR1/HLA-A2 on the surface of U266 cells by flow cytometry as early as 6 and 24 hours after co-culture with soluble NE and P3, respectively. Cross-presentation was also seen in the cells that were co-cultured with irradiated PMN to a similar extent in comparison with the cells that were cultured with soluble NE and P3, however, cross-presentation occurred at an earlier time point (1 hour) in the cells that were cultured with PMNs. Cross-presentation was abrogated by lactacystin, a proteasome inhibitor, and brefeldin A, an ER/Golgi transport inhibitor, indicating that NE and P3 cross-presentation occurs through conventional cross-presentation mechanisms. Furthermore, because immune modulatory drugs and proteasome inhibitors are part of the standard of care therapy for MM, and since both have been shown to affect cross-presentation by DC, we tested whether lenalidomide and bortezomib altered PR1 cross-presentation by U266 and normal DC. U266 and DC were cultured with irradiated PMN in the presence of increasing doses of bortezomib or lenalidomide and then stained with PR1/HLA-A2. Flow-cytometry analysis showed a significant inhibition of PR1-cross-presentation by U266 after addition of bortezomib, but not lenalidomide. PR1 cross-presentation by DC was not affected by either drug. Finally, we tested whether PR1 cross-presentation caused MM cells to become susceptible to PR1-targeting immunotherapies. U266 cells were cultured with NE or P3 for 24 hours, the time point that showed the maximal cross-presentation, followed by addition of 8F4 antibody or co-culture with PR1-CTL. Using calcein AM cytotoxicity assays, we showed dose dependent killing of U266 by 8F4 and PR1-CTL following PR1 cross-presentation (Fig. 2). Together our data show that PR1 is cross-presented by primary MM cells and cell lines. These findings lay the foundation for the future applications of PR1-targeting immunotherapies in MM. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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