MCF 10A Research Articles

Characterization and Cytotoxic Activity of <i>Dalbergia latifolia</i> Wood Extract

<i>Dalbergia latifolia</i> commonly known as the Indian rosewood, commonly found in association with Teak trees population in and around Western Ghat areas of Tamil Nadu and Kerala in India. From the heartwood of roots and trunk many phytochemical contents were investigated. A wide spectrum of flavanoids namely, flavones, isoflavones, neoflavones and chalcones have been obtained. Specifically, one of the flavonoids compound chalcone compound-Isoliquiritigenin (ILTG, 2',4',4'-trihydroxychalcone) shows various pharmacological properties, including cytotoxic activities. This study revealed the cytotoxic effect of Isoliquiritigenin, efficiently controls the invasive capacity of breast cancer cells MCF10A human mammary epithelial cell line <i>through</i> uplifting G1-phase cell cycle, even arrest at low concentration, also accelerate the extracellular signal regulated kinase signaling pathway to topup the proteins combined with apoptosis and arrest cancer. The cytotoxicity <i>invitro</i> of isoliquitrigenin against MCF10A ATCC cells was investigated by MTT assay. Apoptosis was evaluated by microscopy and flow cytometry. Isoliquiritigenin exhibited the most potent anticancer activity against MCF 10A ATCC with an IC 50 of 10.00 µg/ml. The methanolic extract expressed cytotoxic effect with an IC50 value of 20 µg/ml. The compounds were also tested for their toxicity on normal human cell lines-LO2 and were found to be nontoxic. Administering the wood extract approaches may help reduce side effects in patients under conventional chemotherapy. These potential molecule may exerts their co-active effects through various pathways and helpful for the development of novel drugs; also conservation of these important germplasm is mandatory.

Novel Homo-Bivalent and Polyvalent Compounds Based on Ligustrazine and Heterocyclic Ring as Anticancer Agents.

Bivalent and polyvalent inhibitors can be used as antitumor agents. In this experiment, eight ligustrazine dimers and seven ligustrazine tetramers linked by alkane diamine with different lengths of carbon chain lengths were synthesized. After screening their antiproliferation activities against five cancer cell lines, most ligustrazine derivatives showed better cytotoxicity than the ligustrazine monomer. In particular, ligustrazine dimer 8e linked with decane-1,10-diamine exhibited the highest cytotoxicity in FaDu cells with an IC50 (50% inhibiting concentration) value of 1.36 nM. Further mechanism studies suggested that 8e could induce apoptosis of FaDu cells through the depolarization of mitochondrial membrane potential and S-phase cell cycle arrest. Inspired by these results, twenty-seven additional small molecule heterocyclic dimers linked with decane-1,10-diamine and nine cinnamic acid dimers bearing ether chain were synthesized and screened. Most monocyclic and bicyclic aromatic systems showed highly selective anti-proliferation activity to FaDu cells and low toxicity to normal MCF 10A cells. The structure-activity relationship revealed that the two terminal amide bonds and the alkyl linker with a chain length of 8–12 carbon were two important factors to maintain its antitumor activity. In addition, the ADMET calculation predicted that most of the potent compounds had good oral bioavailability.

The hexane fraction of the Moringa oleifera Lam seed extract induces apoptosis, causes cell cycle arrest, and modulates expression of HSP60, NPM, PGK1, RCN1, and PDIA1 in MCF7 cells

Moringa oleifera Lam is a medicinal plant that had been shown to have anticancer activity. Previously, the hexane fraction of the seeds (HF-CEE) was found to inhibit breast cancer (MCF7) cell proliferation at IC50 of 130 μg/ml. However, the mechanism by which HF-CEE inhibits MCF7 cells remained unknown. To elucidate the mechanism, we investigated the effect of HF-CEE on apoptosis, cell cycle, and the cell proteome of MCF7 cells. The HF-CEE was prepared by fractionating the non-polar fraction of the ethanolic extract of M. oleifera seeds using the hexane solvent. The antiproliferative effect of HF-CEE on the growth of MDA-MB-231 cells (another breast cancer cells) was performed using Presto Blue assay. The morphologies of the untreated and treated cells (MCF7, MDA-MB-231 and MCF 10A cells) were observed using inverted light microscope. The apoptotic and cell cycle arrest effects of HF-CEE on MCF7 cells were determined using flow cytometry analyses. 2-DE proteomics analysis was employed to identify the protein biomarkers of MCF7 cells that have their expression levels altered by HF-CEE treatment. qRT-PCR method was used to validate the 2-DE results. The gene ontology analysis was carried out using PANTHER server to identify the mechanisms and biological processes that are dependent on the functions of the identified biomarkers.Our results showed that HF-CEE selectively inhibited the proliferation of the breast cancer cells (MCF7 and MDA-MB-231) but not the MCF 10A non-tumor cells. MCF7 and MDA-MB-231 cells treated with HF-CEE showed morphological properties of dead cells, but MCF 10A cells did not. HF-CEE also induced apoptosis and cell cycle arrest at the S and G2/M phases in MCF7 cells. 2-DE analysis revealed that HF-CEE altered the regulation of several cancer-related proteins in MCF7 cells, such as heat shock protein 60 (HSP60), nucleophosmin (NPM), protein disulphide isomerase (PDIA1), phosphoglycerate kinase 1 (PGK1), and reticulocalbin 1 (RCN1). In conclusion, HF-CEE induced cell death in MCF7 cells via apoptosis, cell cycle arrest, and other mechanisms such as the alteration of the expression of proteins that are involved in glycolysis, cancer invasiveness and metastasis among others. Results of this study provide further insights into the mechanisms that underlie the antiproliferative effect of HF-CEE on MCF7 cells.

The response of phyllodes tumor of the breast to anticancer therapy: An in vitro and ex vivo study.

Phyllodes tumors of the breast (PTB) are uncommon stromal-epithelial neoplasms, with the main recommended treatment being surgical removal. However, even with adequate resection, the risk of recurrence in the malignant form remains as high as 40%, and there is no recognized consensus on the most effective drugs for PTB. In the present study, an ex vivo model of malignant phyllodes and derived primary cell cultures were used to evaluate the effectiveness of a panel of different drugs, including the Bcl-2/Bcl-xL inhibitor ABT-263, salinomycin (SAL), doxorubicin (DOX), paclitaxel (TAX), vincristine (VCR), colchicine (COL) and cisplatin (CIS). ABT-263, SAL and DOX were highly effective towards phyllodes spindle cells when assessed in the ex vivo model, contributing to ~98% tumor cell death. Furthermore, ABT-263 was highly selective for tumor cells in this system, and exhibited little toxic effect on adjacent normal epithelial cells. Furthermore, consistent with findings in the ex vivo model, ABT-263 was significantly less toxic towards MCF 10A non-tumorigenic breast epithelial cells compared with SAL and DOX. A conditional reprogramming strategy was subsequently used, involving Rho kinase inhibition, to successfully generate primary phyllodes tumor cells that could be cultured for several passages. The primary cells were sensitive to DOX with an IC50 of 0.40±0.07 µM in a standard viability assay and the preliminary results were obtained indicating sensitivity to ABT-263 and SAL. The present study demonstrated the feasibility of using explants and primary cells for drug discovery, selectively targeting PTB cells.

MicroRNA-155-3p promotes breast cancer progression through down-regulating CADM1.

Background/purposeCell adhesion molecule 1 (CADM1) functions as a tumor suppressor and has been identified to be frequently inactivated in breast cancer, and closely associated with patients’ poor prognosis and advanced TNM stage. However, the mechanisms underlying CADM1 in breast cancer progression remains incompletely clear. miR-155, a predicted modulator of CADM1 was reported to be overexpressed in breast cancer, and its high expression level was closely related to the malignant progression of breast cancer. The present study aimed to explore whether miR-155-3p could modulate CADM1 expression and then involved in the progression of breast cancer.MethodsThe expression patterns of miR-155-3p in breast cancer tissues and cell lines were determined by RT-PCR technology. The relationship between CADM1 and miR-155-3p were determined by the luciferase gene reporter and Western Blot (WB) assays. Cell proliferation, apoptosis rates and tumorigenesis were determined by CCK-8, flow cytometry and in vivo xenotransplanation experiments, respectively.ResultsmiR-155-3p was up-regulated in breast cancer tissues and cells when compared to the adjacent normal tissues and normal breast MCF 10A cells. The mRNA and protein levels of CADM1 showed opposite expression patterns to that of miR-155-3p expression detected, and miR-155-3p could negatively regulate CADM1 expression in breast cancer MCF-7 cells. Moreover, gain-of function assay showed that overexpression of miR-155-3p promoted cell proliferation, tumorigenesis and repressed cell apoptosis, but these effects were all significantly impaired when the cells were simultaneously transfected with OE-CADM1, the overexpressing vector of CADM1.ConclusionThis study revealed that miR-155-3p could accelerate the progression of breast cancer via down-regulation of CADM1 expression.

Cancer Cells Sense Fibers by Coiling on them in a Curvature-Dependent Manner.

SummaryMetastatic cancer cells sense the complex and heterogeneous fibrous extracellular matrix (ECM) by formation of protrusions, and our knowledge of how cells physically recognize these fibers remains in its infancy. Here, using suspended ECM-mimicking isodiameter fibers ranging from 135 to 1,000 nm, we show that metastatic breast cancer cells sense fiber diameters differentially by coiling (wrapping-around) on them in a curvature-dependent manner, whereas non-tumorigenic cells exhibit diminished coiling. We report that coiling occurs at the tip of growing protrusions and the coil width and coiling rate increase in a curvature-dependent manner, but time to maximum coil width occurs biphasically. Interestingly, bundles of 135-nm diameter fibers recover coiling width and rate on 1,000-nm-diameter fibers. Coiling also coincides with curvature-dependent persistent and ballistic transport of endogenous granules inside the protrusions. Altogether, our results lay the groundwork to link biophysical sensing with biological signaling to quantitate pro- and anti-invasive fibrous environments.Video

Folic acid decorated magnetic nanosponge: An efficient nanosystem for targeted curcumin delivery and magnetic resonance imaging

Magnetic drug delivery system is one of the most important strategies for cancer diagnosis and treatment. In this study, a novel theranostic system was fabricated based on cyclodextrin nanosponge (CDNS) polymer anchored on the surface of Magnetite nanoparticles (Fe3O4/CDNS NPs) which was then decorated with folic acid (FA) as a targeting agent (Fe3O4/CDNS-FA). Curcumin (CUR), a hydrophobic model drug, was next loaded into the cyclodextrin cavity and polymeric matrix of CDNS (Fe3O4/CDNS-FA@CUR). The system was fully characterized. The in vitro release study revealed pH-sensitive behavior. Cytotoxicity assays indicated a negligible toxicity for CUR free Fe3O4/CDNS-FA NPs against both of M109 cancerous cells and MCF 10A normal cells. CUR-loaded Fe3O4/CDNS-FA NPs exhibited higher toxicity against M109 cancerous cells than MCF 10A normal cells (p < 0.05). Fe3O4/CDNS-FA@CUR NPs resulted in much more cell viability on normal cells than pure CUR (p < 0.05). Moreover, blood compatibility study showed minor hemolytic activity. In vitro MRI studies illustrated negative signal increase in cells affirming acceptable diagnostic ability of the nanocarrier. The T2 MR signal intensity for Fe3O4/CDNS-FA@CUR NPs in M109 cells was around 2-fold higher than that of MCF 10A cells. This implies two times higher selective cellular uptake of the Fe3O4/CDNS-FA@CUR NPs into M109 cell compared to MCF 10A. The multifunctional nanocarrier could be considered as promising candidate for cancer theranostics because of the smart drug release, selective cytotoxicity, suitable hemocompatibility, and proper T2 MRI contrast efficiency.

Enzymatic rhamnosylation of anticancer drugs by an α-L-rhamnosidase from Alternaria sp. L1 for cancer-targeting and enzyme-activated prodrug therapy.

The synthesis of rhamnosylated compounds has gained great importance since these compounds have potential therapeutic applications. The enzymatic approaches for glycosylation of bioactive molecules have been well developed; however, the enzymatic rhamnosylation has been largely hindered by lacking of the glycosyl donor for rhamnosyltransferases. Here, we employed an α-L-rhamnosidase from Alternaria sp. L1 (RhaL1) to perform one-step rhamnosylation of anticancer drugs, including 2'-deoxy-5-fluorouridine (FUDR), cytosine arabinoside (Ara C), and hydroxyurea (Hydrea). The key synthesis conditions including substrate concentrations and reaction time were carefully optimized, and the maximum yields of each rhamnosylated drugs were 57.7 mmol for rhamnosylated Ara C, 68.6 mmol for rhamnosylated Hydrea, and 42.2 mmol for rhamnosylated FUDR. It is worth pointing out that these rhamnosylated drugs exhibit little cytotoxic effects on cancer cells, but could efficiently restore cytotoxic activity when incubated with exogenous α-L-rhamnosidase, suggesting their potential applications in the enzyme-activated prodrug system. To evaluate the cancer-targeting ability of rhamnose moiety, the rhamnose-conjugated fluorescence dye rhodamine B (Rha-RhB) was constructed. The fluorescence probe Rha-RhB displayed much higher cell affinity and cellular internalization rate of oral cancer cell KB and breast cancer cell MDA-MB-231 than that of the normal epithelial cells MCF 10A, suggesting that the rhamnose moiety could mediate the specific internalization of rhamnosylated compounds into cancer cells, which greatly facilitated their applications for cancer-targeting drug delivery.

Structure-Antioxidant-Antiproliferative Activity Relationships of Natural C7 and C7-C8 Hydroxylated Flavones and Flavanones.

Common food flavonoids: chrysin, apigenin, luteolin, diosmetin, pinocembrin, naringenin, eriodictyol, hesperetin, and their analogues with an additional hydroxyl group at the C-8 position obtained via biotransformation were tested for antioxidant activity using the ABTS, DPPH, and ferric ion reducing antioxidant power (FRAP) methods. They were also tested for antiproliferative activity against selected human cancer cell lines—MV-4-11 (biphenotypic B myelomonocytic leukemia), MCF7 (breast carcinoma), LoVo (colon cancer), LoVo/DX (colon cancer doxorubicin resistant), and DU 145 (prostate cancer)—and two normal human cell lines—MCF 10A (breast cells) and HLMEC (lung microvascular endothelial cells). Flavonoids with a C7–C8 catechol moiety indicated much higher antioxidant activity compared with the C7 hydroxy analogues. However, because they were unstable under the assay conditions, they did not show antiproliferative activity or it was very low.

Abstract 2346: Curcumin suppresses PhIP induced inflammatory protein expression in breast epithelial cells

Abstract PhIP (2-Amino-1-methyl-6-phenylimidazo [4,5-b] pyridine), a prominent mutagen/carcinogen is a heterocyclic amine found in meat cooked at high temperature and in tobacco. PhIP is known to induce liver, colon, skin, prostate, and breast cancer as evident from several in vivo and in vitro studies. Our previous studies have demonstrated that curcumin significantly inhibits PhIP-induced DNA adduct formation and DNA double-strand breaks with the concomitant decrease in reactive oxygen species (ROS) production via multiple molecular targets. We hypothesize that PhIP induced ROS production induces inflammatory proteins and co-treatment with curcumin would suppress the induced inflammation. Therefore, human breast epithelial cells (MCF 10A) were used to test the expression of inflammatory proteins. MCF 10A cells were treated with PhIP alone and in presence of curcumin. Untreated cells and treated only with curcumin served as control. Human Inflammation array (Ray Biotech) was used to analyze the expression of forty inflammatory proteins. Results show that the expression of twenty inflammatory proteins is induced in MCF 10 A cells when treated with PhIP as compared to the controls. Presence of curcumin reduces the induced expression of these twenty inflammatory proteins. However, three inflammatory proteins (IL-8, IL 12p40 and ICAM-1) showed a significantly higher level of expression with PhIP and curcumin co-treated cells as compared to cells treated with PhIP alone. It is shown that higher expression of IL-8, IL 12p-40 and ICAM-1 is involved in chemotactic factor involve in attracting the neutrophils, basophils; activation of T and NK cells which might be helping in coordinating the inflammation process. Hence the present study suggests that curcumin is a promising natural compound to revert the effect of PhIP induced inflammation. Citation Format: Ashok Jain. Curcumin suppresses PhIP induced inflammatory protein expression in breast epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2346.

Synergistic effect of granzyme B-azurin fusion protein on breast cancer cells.

As one of the most prevalent malignancies, breast cancer still remains a significant risk for public health. Common therapeutic strategies include invasive surgery, chemotherapy and anti-herceptin antibodies. Adverse effects, drug resistance and low efficacy of current therapies necessitates the emergence of more effective platforms. Naturally released by the immune system, granzyme B activates multiple pro-apoptotic pathways by cleaving critical substrates. Bacterial cupredoxin, azurin, selectively targets cancer cells via a p53-dependent pathway. Fused by a linker, GrB-Azurin fusion protein was overexpressed in HEK293T cells, and purified by metal chromatography. SDS-PAGE, Western blotting and ELISA were performed to confirm successful expression, purification and analyze binding properties of the fusion protein. After treatment of various breast cancer cell lines with increasing concentrations of GrB-Azurin, quantitative real-time RT-PCR was used to measure relative expression of p21, Fas and DR5 pro-apoptotic genes. The results of DNA fragmentation and WST-1 cell viability assays indicated significant apoptosis induction in MDA-MB-231, MCF7 and SK-BR-3 cells, while insignificant cytotoxicity was detected on MCF 10A normal breast cells. Herein, we report the development of a novel biotherapeutic against breast cancer. Selective effectiveness of GrB-Azurin fusion protein on different breast cancer cells highlighted the potential of the designed construct as a candidate anti-cancer biodrug.

Corrigendum] Antiproliferative effects of imatinib mesylate on ZR‑75‑1 and MDA‑MB‑231cell lines via PDGFR‑β, PDGF‑BB, c‑Kit and SCF expression.

An interested reader drew to our attention that the above study appeared to contain a high level of overlap with an article by the same authors published in the journal Drug Design, Development and Therapy [Kadivar A, KamalidehghanB, Akbari JavarH, Karimi B, Sedghi R and NoordinMI: Antiproliferation effect of imatinib mesylate on MCF7, T‑47D tumorigenic and MCF10A nontumorigenic breast cell lines via PDGFR‑β, PDGF‑BB, c‑Kit and SCF genes. Drug Des Devel Ther11: 469‑481, 2017]. Following an internal investigation and also in liaison with the authors, it was established that, although the studies were conducted along broadly similar lines, the papers contained entirely different data involving two different subsets of cell lines; the submission to Drug Des Devel Ther aimed to explore the effects of imatinib mesylate on three different groups, with each group being represented by a cell line, whereas the submission to IntJ Mol Med explored the effectiveness of imatinib mesylate in breast cancer cell lines. In spite of this, considering the relatedness of the articles and the fact that the paper to Drug Des Devel Ther was submitted first and published while the Int J Mol Med paper was passing through the peer‑review process, the authors concede that they should have properly referenced their paper submitted to Drug Des Devel Ther in the Int J Mol Med paper. Note that the publishers of Drug Des Devel Ther, with whom we were liaising, agreed with the decision to issue a Corrigendum for this paper that acknowledges the article published in Drug Des Devel Ther. The authors regret their failure to acknowledge the related paper in this instance, and apologize to the readership for this oversight. [the original article was published in International Journal of Molecular Medicine 14: 414‑424, 2018; DOI: 10.3892/ijmm.2018.3590].

Green synthesis of gold nanoparticles from Dunaliella salina, its characterization and in vitro anticancer activity on breast cancer cell line

An ecofriendly and efficient method (photoinduced) has been used for green synthesis of stable gold nanoparticles (AuNPs) using aqueous extract as a reducing and stabilizing agent, derived from halotolerant microalga Dunaliella salina. Synthesis of AuNPs was confirmed by UV–Vis spectroscopy showing sharp SPR band at 560 nm after 30 min exposure of sunlight. The synthesis was optimized further as exposure(75min) to sunlight, aqueous extract of Dunaliella (AED) inoculum dose(30%) and HAuCl4.xH2O (1 mM). The presence of nearly spherical shape of AuNPs with average size of 22.4 nm and crystalline nature were confirmed by TEM, SAED and XRD analysis. The XPS analysis of AuNPs showed presence of two individual peaks at 85.17 and 88.94eV that attributed to Au 4f7/2 and Au 4f5/2 respectively. Phycochemical analysis of AED demonstrated presence of phenolics, flavonoids, tannin and proteins. AED when subjected to SDS-PAGE showed protein bands of 92 and 66 KDa. AuNPs were tested for anticancer activity on MCF7 (cancer) and MCF 10A (normal) cell lines, keeping commercial drug cisplatin as positive control. AuNPs selectively killed cancer cells and were not detrimental to the normal cell line whereas cisplatin killed normal cells also at 48 h exposure, therefore, such AuNPs may be used as an anticancer agent.

Improved curcumin loading, release, solubility and toxicity by tuning the molar ratio of cross-linker to β-cyclodextrin

A novel β-cyclodextrin-based nanosponge (CDNS) was proposed as curcumin (CUR) delivery system improving pharmacokinetics and anticancer activity of CUR. The effect of molar ratio of Epiclon (EPI) as cross-linker and β-cyclodextrin (βCD) on the porosity, surface area, swelling ratio, CUR solubility and loading capacity, rate of drug release and selective toxicity of the CDNSs was fully investigated. The high degree of cross-linking led to the formation of mesoporous CDNS having high specific surface area and high loading capacity. All CUR-free CDNSs showed no toxicity against MCF 10A and 4T1 cells as normal and cancerous cells, respectively. While CDNSs-CUR exhibited selective toxicity against cancerous cells. In sum, high CUR aqueous solubility, significant loading and controllable release of the CUR, outstanding and selective toxicity against cancerous cells make CDNS8-CUR (EPI/βCD = 8) as promising candidate for further study in the cancer therapy.

High-sensitivity detection of metastatic breast cancer cells via terahertz chemical microscopy using aptamers

We demonstrate high-sensitivity detection of metastatic breast cancer cells using terahertz (THz) chemical microscopy (TCM) with aptamers as ligands. For the aptamers, we use previously developed synthetic single stranded (ss) DNA aptamers mammaglobin B1 (MAMB1) and mammaglobin A2 (MAMA2) that bind to mammaglobin B and mammaglobin A proteins, respectively, which are overexpressed on the surface of MCF7 and MDA-MB-415 breast cancer cells. Each aptamer was immobilized on the surface of a sensing plate, and the amplitude of the THz signal was measured upon the binding of each aptamer to different number (10–106) of its target breast cancer cells. A change in the THz amplitude was observed when MAMB1 and MAMA2 bind to MCF7 and MDA-MB-415, respectively. We find that this change was linear as a function of the log number of breast cancer cells used. No change in the THz amplitude was observed when the same number of normal breast cells (MCF 10A) were used. Moreover, MAMB1 and MAMA2 did not show binding to the counter breast cancer cells, indicating high selectivity. We have demonstrated that the TCM using aptamers as ligands has a limit of detection as small as 1 breast cancer cell in 100 μL of sample. Results described here indicate that the TCM could be a powerful tool to detect metastatic breast cancer cells.

Valtrate from Valeriana jatamansi Jones induces apoptosis and inhibits migration of human breast cancer cells in vitro

Valtrate is a principle compound isolated from Valeriana jatamansi Jones, a traditional Chinese folk medicine originally used to treat various nervous disorders. Here, we found that valtrate exhibited significant anti-cancer activity in vitro, especially in human breast cancer cells, while displayed relatively low cytotoxicity to normal human breast epithelial cells (MCF 10A). Valtrate induced cell cycle arrest at G2/M stage and apoptosis in MDA-MB-231 and MCF-7 cells, with reduced expression of p-Akt (Ser 473), cyclin B1 and caspase 8, and increased expression of p21, p-cdc2, cleaved-caspase 3, cleaved-caspase 7 and poly (ADP-ribose) polymerase (PARP). In addition, valtrate inhibited cell migration through down-regulation of MMP-9 and MMP-2 expression. These results demonstrate that valtrate possesses anti-breast cancer activities via cell cycle arrest, apoptosis, and inhibition of cell migration, thus supporting valtrate as a potential antitumor agent.

Highly Cancer Selective Antiproliferative Activity of Natural Prenylated Flavonoids.

Xanthohumol (XN) and four minor hops prenylflavonoids: α,β-dihydroxanthohumol (2HXN), isoxanthohumol (IXN), 8-prenylnaringenin (8PN), and 6-prenylnaringenin (6PN), were tested for antiproliferative activity towards human cancer and normal cell lines. Nonprenylated naringenin (NG) was used as a model compound. Xanthohumol, α,β-dihydroxanthohumol and 6-prenylnaringenin were the most active compounds. Xanthohumol exhibited higher antiproliferative activity than cisplatin (CP) against five cancer cell lines: ovarian resistant to cisplatin A2780cis, breast MDA-MB-231 and T-47D, prostate PC-3, and colon HT-29. Isoxanthohumol was more potent than cisplatin against breast cancer cell lines MDA-MB-231 and T-47D whereas 6-prenylnaringenin was stronger than cisplatin against breast cancer cell line T-47D. It was found that tested chalcones possessed highly selective antiproliferative activity towards all tested breast cancer lines compared to the normal breast MCF 10A cell line (the calculated selectivity index ranged from 5 to 10). Low antiproliferative activity of naringenin indicates the importance of the prenyl group with respect to antiproliferative activity.

DIFFERENTIAL EFFECT OF PURIFIED QUERCETIN AND ITS DERIVATIVES FROM IN VITRO CELL SUSPENSION CULTURES OF CAESALPINIA PULCHERRIMA SW. AGAINST SELECTED CANCER CELL LINES AND ITS MODE OF ACTION

Tribal people use the floral extract of Caesalpinia pulcherrima to cure liver, stomach and skin prone disorders in traditional Indian medicine. This study aimed to evaluate the effect of purified quercetin and its derivatives from in vitro cell suspension cultures of C. pulcherrima Sw. against SW 480, HeLa, MCF-7 and MCF 10A cell lines and its mode of action. Standard protocol was developed for callus induction using leaf explants. Cytotoxic effect was evaluated against SW 480, HeLa, MCF-7 and MCF 10A cells by MTT assay. Apoptosis was evaluated via Hoechst analysis, flow cytometry, mitochondrial membrane potential and caspase 3 and 9 expression. 2, 4-D (2.5 mg/l), BAP (2.5 mg/l) + kin (1 mg/ml) was effective for remarkable callus induction. Further, cell suspension culture was established. Effect of elicitors on cell suspension culture was also carried. Sucrose, ABA and salicylic acid (SA) at different concentrations influenced cell biomass and quercetin synthesis. Cells cultured on the medium fortified with 45 g/L sucrose without ABA/SA showed the highest quercetin content (16.5 mg/g). Quercetin was purified, fractionated by HPLC-DAD and was further analyzed by NMR revealed a major fraction of quercetin (3, 5, 7, 3’, 4’-pentahydroxyflavon). Insignificant cytotoxicity was noticed in SW 480, HeLa, MCF 10A when compared to MCF-7 cell lines exposed to different concentrations of purified quercetin for 24- 48 h. Similarly, the apoptosis by nuclei staining using Hoechst 33258 revealed a concentration dependent effect on MCF 7 cells only. This was further substantiated by caspase-9 and 3 induction and mitochondrial depolarization as revealed by flow cytometry. Overall, the results showed that quercetin and its derivatives induced effective apoptosis on MCF-7 cells. Quercetin isolated from the in vitro cell suspension culture of C. pulcherrima showed significant cytotoxicity and apoptotic activity towards MCF-7 cell lines as compared to other cell lines.&#x0D; Keywords: Caesalpinia pulcherrima; quercetins; suspension culture; cytotoxicity; apoptotic.

Antitumor potential of Pllans–II, an acidic Asp49–PLA2 from Porthidium lansbergii lansbergii snake venom on human cervical carcinoma HeLa cells

This work shows for the first time the antitumoral and angiostatic potential effects of Pllans–II, an acidic monomeric Asp49–PLA2 from Porthidium lansbergii lansbergii snake venom on HeLa cells. Pllans–II exhibited a predominant dose–dependent cytotoxicity on cervical carcinoma HeLa cells more than human breast cancer MCF7 and MDA–MB–231 cells. Interestingly, Pllans–II showed negligible effect in vitro on normal cells such as MCF 10A, a non–tumorigenic breast epithelial cell and endothelial cells–HUVEC. Besides, Pllans–II induced a significative cell cycle arrest in G1 phase of treated HeLa. Flow Cytometry analysis showed that Pllans–II induced both early and late apoptosis on HeLa cells, verified by the modulation of important genes mediators of apoptosis pathways such as CASP8, BCL2L1, BCL2, BAX, BAD and BIRC5, showing a possible apoptotic cell death triggered through extrinsic pathways. Pllans–II was also able to inhibit cell migration on HeLa cells by interfering with α5 and β1–containing integrins. In addition, Pllans–II inhibited in vitro angiogenesis on endothelial HUVEC cells through VEGF–independent pathway. Our results display that Pllans–II is able to be an anticancer target for the development of a new antitumoral prototype.

Structure-activity relationship and pharmacokinetic studies of 3-O-substitutedflavonols as anti-prostate cancer agents

Thirty-eight 3-O-substituted-3′,4′-dimethoxyflavonols and twenty-five 3-O-substituted-3′,4′,7-trimethoxyflavonols have been synthesized for systematic investigation on the structure-activity relationships of 3-O-substituted-3′,4′-dimethoxyflavonols in three human prostate cancer cell models. Our findings indicate that incorporation of an appropriate amino group to 3-OH of 3′,4′-dimethoxyflavonol and 3′,4′,7-trimethoxyflavonol through a 3- to 5-carbon linker can substantially improve the in vitro antiproliferative potency in three human prostate cancer cell models, but not in two non-neoplastic human epithelial cell models (MCF 10A and PWR-1E). 1-Methylpiperazine, pyrrolidine, and dibutylamine are optimal terminal amine groups that, in combination with a 3- to 5-carbon linker, are notably beneficial to the anti-proliferative potency of 3-O-substituted-3′,4′-dimethoxyflavonols. It is worth noting that 3-O-(4-methylpiperazin-1-yl)propyl-3′,4′,7-trimethoxyflavonol (76) induces PC-3 cell death in a completely different way from 3-O-pyrrolidinopentyl-3′,4′,7-trimethoxyflavonol (81) even though they belong to 3-O-substituted-3′,4′,7-trimethoxyflavonols and exhibit similar potency in inhibiting PC-3 cell proliferation, suggesting that the mechanism of action for each specific 3-O-substitutedflavonol varies with different amino moiety. 3-O-(N,N-Dibutylamino)propyl-3′,4′-dimethoxyflavonol (42) emerged as the most promising derivative due to its substantially improved potency in cell models, superior bioavailability in rats, and good selectivity of inhibiting prostate cancer cell proliferation over non-neoplastic human epithelial cell proliferation.