Anticancer Constituents Research Articles

Medicinal plants combating against cancer--a green anticancer approach.

Cancer is the most deadly disease that causes the serious health problems, physical disabilities, mortalities, and morbidities around the world. It is the second leading cause of death all over the world. Although great advancement have been made in the treatment of cancer progression, still significant deficiencies and room for improvement remain. Chemotherapy produced a number of undesired and toxic side effects. Natural therapies, such as the use of plant-derived products in the treatment of cancer, may reduce adverse and toxic side effects. However, many plants exist that have shown very promising anticancer activities in vitro and in vivo but their active anticancer principle have yet to be evaluated. Combined efforts of botanist, pharmacologist and chemists are required to find new lead anticancer constituent to fight disease. This review will help researchers in the finding of new bioactive molecules as it will focus on various plants evaluated for anticancer properties in vitro and in vivo.

Lappaol F, a Novel Anticancer Agent Isolated from PlantArctium LappaL.

In an effort to search for new cancer-fighting therapeutics, we identified a novel anticancer constituent, Lappaol F, from plant Arctium Lappa L. Lappaol F suppressed cancer cell growth in a time- and dose-dependent manner in human cancer cell lines of various tissue types. We found that Lappaol F induced G(1) and G(2) cell-cycle arrest, which was associated with strong induction of p21 and p27 and reduction of cyclin B1 and cyclin-dependent kinase 1 (CDK1). Depletion of p21 via genetic knockout or short hairpin RNA (shRNA) approaches significantly abrogated Lappaol F-mediated G(2) arrest and CDK1 and cyclin B1 suppression. These results suggest that p21 seems to play a crucial role in Lappaol F-mediated regulation of CDK1 and cyclin B1 and G(2) arrest. Lappaol F-mediated p21 induction was found to occur at the mRNA level and involved p21 promoter activation. Lappaol F was also found to induce cell death in several cancer cell lines and to activate caspases. In contrast with its strong growth inhibitory effects on tumor cells, Lappaol F had minimal cytotoxic effects on nontumorigenic epithelial cells tested. Importantly, our data also demonstrate that Lappaol F exhibited strong growth inhibition of xenograft tumors in nude mice. Lappaol F was well tolerated in treated animals without significant toxicity. Taken together, our results, for the first time, demonstrate that Lappaol F exhibits antitumor activity in vitro and in vivo and has strong potential to be developed as an anticancer therapeutic.

Cytotoxic and potential anticancer constituents from the stems of Schisandra pubescens

Context: The diethyl ether extract of the stems of Schisandra pubescens Hemsl. et Wils. (Schisandraceae) was found to exhibit cytotoxic activity in vitro. However, investigations of the bioactive constituents of this plant have been very limited.Objective: Elucidation of the cytotoxic constituents of S. pubescens was performed.Methods: Repeated silica gel column chromatography and preparative TLC were used for the chemical investigation of the diethyl ether extract of S. pubescens stems. All isolates were evaluated for their in vitro cytotoxicity against A549, PC-3, KB and KBvin human cancer cell lines.Results: Nine known compounds were obtained, including four lignans, epischisandrone (1), tigloylgomisin P (2), cagayanone (3) and (−)-gomisin L2 (4), together with five triterpenoids, micranoic acid B (5), lancifodilactone H (6), coccinic acid (7), schisanlactone B (8) and anwuweizonic acid (9). Compounds 2–6 and 8 showed moderate to marginal cytotoxicity, with GI50 values of 11.83–35.65 μM.Conclusion: The isolation of 1–9 from S. pubescens and the cytotoxicities of 3–6 are first reported. Compounds 2–6 and 8 could be the active principles responsible for the anticancer effects of S. pubescens.

Cytotoxic activity of marine algae against cancerous cells

This paper presents an investigation on the cytotoxic activity in human tumor cell from dichloromethane, chloroform, methanol, ethanol, water extracts, and hexane and chloroform fractions from green, brown and red algae collected at Riacho Doce Beach, north coast of Alagoas, Brazil, against the cancer cells K562 (chronic myelocytic leukemia), HEp-2 (laryngeal epidermoid carcinoma) and NCI-H292 (human lung mucoepidermoid carcinoma) through the MTT colorimetric method. The dichloromethane extract and chloroform fraction of Hypnea musciformis showed the best cytotoxic activity against K562 (3.8±0.2 μg.mL-1 and 6.4±0.4 μg.mL-1, respectively). Dichloromethane extracts of Dictyota dichotoma (16.3±0.3 μg.mL-1) and the chloroform fraction of H. musciformis (6.0±0.03 μg.mL-1) and chloroform fraction of P. gymnospora (8.2±0.4) were more active against HEp-2 as well as ethanol extracts of P. gymnospora (15.9±2.8 μg.mL-1) and chloroform fraction of H. musciformis (15.0±1.3 μg.mL-1) against the cell NCI-H292. The constituents with higher anticancer action are present in the extracts of dichloromethane and chloroform and in the chloroform fraction of H. musciformis, Digenea simplex, P. gymnospora, and D.dichotoma. In the case of the seaweed S. vulgare, the anticancer constituents are present in the aqueous extract.

Autophagy Fails to Alter Withaferin A-Mediated Lethality in Human Breast Cancer Cells

We have shown previously that withaferin A (WA), which is a highly promising anticancer constituent of Ayurvedic medicine plant Withania somnifera, inhibits viability of cultured breast cancer cells in association with reactive oxygen species (ROS)-dependent apoptosis induction. Because ROS production is implicated in induction of autophagy, which is an evolutionary conserved process for bulk degradation of cellular components including organelles (e.g., mitochondria) and considered a valid cancer chemotherapeutic target, we questioned whether WA treatment resulted in autophagy induction. Indeed exposure of MDA-MB-231 and MCF-7 human breast cancer cells as well as a spontaneously immortalized and non-tumorigenic normal human mammary epithelial cell line (MCF-10A) to pharmacologic concentration of WA resulted in autophagy as evidenced by transmission electron microscopy, processing of microtubuleassociated protein 1 light chain 3 isoform B, and/or acridine orange staining. Inhibition of MDA-MB-231 xenograft growth in vivo by WA administration was also associated with a significant increase in level of LC3 protein in the tumor. However, WA-mediated inhibition of MDA-MB-231 and MCF-7 cell viability was not compromised either by pharmacological suppression of autophagy using 3-methyl adenine or genetic repression of autophagy by RNA interference of Atg5, a critical component of the autophagic machinery. Finally, Beclin1 was dispensable for WA-mediated autophagy as well as inhibition of MDA-MB-231 cell viability. Based on these observations we conclude that autophagy induction fails to have any meaningful impact on WA-mediated lethality in breast cancer cells, which may be a therapeutic advantage because autophagy serves to protect against apoptosis by several anticancer agents.

Synergistic anticancer effects of curcumin and resveratrol in Hepa1-6 hepatocellular carcinoma cells

Hepatocellular carcinoma remains one of the most prevalent malignancies worldwide. Curcuma aromatica and Polygonum cuspidatum are one of the commonly used paired-herbs for liver cancer treatment. Curcumin and resveratrol are the major anticancer constituents of Curcuma aromatica and Polygonum cuspidatum, respectively. Curcumin and resveratrol have been found to exhibit a synergistic anticancer effect in colon cancer. However, the combined effect of curcumin and resveratrol against hepatocellular carcinoma remains unknown. In the present study, we evaluated the combined effects of curcumin and resveratrol in hepatocellular carcinoma Hepa1-6 cells. The results showed that curcumin and resveratrol significantly inhibited the proliferation of Hepa1-6 cells in a dose- and time-dependent manner. The combination treatment of curcumin and resveratrol elicited a synergistic antiproliferative effect in Hepa1-6 cells. The apoptosis of Hepa1-6 cells induced by the combination treatment with curcumin and resveratrol was accompanied by caspase-3, -8 and -9 activation, which was completely abrogated by a pan caspase inhibitor, Z-VAD-FMK. Combination of curcumin and resveratrol upregulated intracellular reactive oxygen species (ROS) levels in Hepa1-6 cells. The ROS scavenger, NAC, partially attenuated the apoptosis and caspase activation induced by the combination treatment of curcumin and resveratrol. In addition, the combination of curcumin and resveratrol downregulated XIAP and survivin expression. These data suggest that the combination treatment of curcumin and resveratrol is a promising novel anticancer strategy for liver cancer. The present study also provides new insights into the effective mechanism of paired-herbs in traditional Chinese medicine.

Anti-inflammatory and anticancer activities of extracts and compounds from the mushroom Inonotus obliquus

Mushroom Inonotus obliquus (I. obliquus) has been used as functional food and traditional Chinese herbs for long time. An efficient method for bioassay-guided preparative isolation was used for identifying the anti-inflammatory and anticancer constituents in I. obliquus. The petroleum ether and ethyl acetate fractions were found to have significant inhibition effects on NO production and NF-κB luciferase activity in macrophage RAW 264.7 cells and cytotoxicity against human prostatic carcinoma cell PC3 and breast carcinoma cell MDA-MB-231. Six main constituents were isolated from these two fractions and they were identified as lanosterol (1), 3β-hydroxy-8,24-dien-21-al (2), ergosterol (3), inotodiol (4), ergosterol peroxide (5) and trametenolic acid (6). Compound ergosterol, ergosterol peroxide and trametenolic acid showed anti-inflammatory activities and ergosterol peroxide and trametenolic acid showed obviously cytotoxicity on human prostatic carcinoma cell PC3 and breast carcinoma MDA-MB-231 cell. The results obtained in this work might contribute to understanding the biological activity of mushroom I. obliquus for food and drug application.

Anticancer Constituents and Cytotoxic Activity of Methanol-Water Extract of <em>Polygonum Bistorta</em> L.

This study was specifically designed to identify anticancer constituents in methanol-water extract of Polygonum bistorta L. and evaluate its cytotoxicity. For this purpose methanol-water (40:60 v/v) extract was subjected to conventional preparative high pressure liquid chromatography and 13 fractions were obtained. Constituents of obtained fractions were separated and identified with the help of GC-MS and LC-DAD-ESI-MS. Anticancer phenolic compounds such as gallic acid, protocatechuic acid, p-hydroxybenzoic acid, chlorogenic acid, vanillic acid, syringic acid, catechol, 4-methyl catechol, syringol and pyrogallol and fatty acids such as linoleic acid, myristic acid and palmitic acid were separated from different fractions. Fractions were evaluated for their cytotoxic activity on a rarely studied human hepatocellular carcinoma cell line (HCCLM3). 11 fractions showed good to strong cytotoxicity in a range of 200 µg/mL-800 µg/mL, whereas 2 fractions did not show any activity even at 800 µg/mL and no anticancer constituent was detected from them. 50 percent growth inhibition (GI50) values for five most active fractions were calculated and results were in a range of 86.5 (±3) µg/mL-126.8 (±3) µg/mL. 3 out of these 5 most active fractions were found to contain phenolic content in them whereas all other fractions containing phenolic content did possess cytotoxic activity that may suggest the importance of phenolic constituents in anticancer activity. Moreover, the results also showed a definite dose dependent relationship between amount of fractions and cytotoxic activity.

Artabotryside A, a constituent from Descurainia sophia (L.) induces cell death in U87 glioma cells through apoptosis and cell cycle arrest at G2/M phase

Descurainia sophia (L.) commonly known as Webb ex Prantl (flixweed) is a Chinese traditional medicinal plant. Seeds of flixweed are used in the treatment of some cancers. However, the anticancer constituents from the seeds have not been investigated. The present study was aimed to identify bioactive compound with anti-glioma activity from the seeds of D. sophia (L.) and to examine the growth inhibitory effect on U87 glioblastoma cells and possible mechanism of action. From bioactive fraction, a flavonol glycoside, artabotryside A, has been isolated and evaluated for in vitro cytotoxicity against U87 glioma cells by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and live/dead assay. Cellular and nuclear morphological changes were observed by phase contrast and fluorescence microscopy, respectively. Cell cycle analysis, apoptotic rate, mitochondrial membrane potential and level of reactive oxygen species (ROS) were detected by flow cytometry. Our results demonstrated that artabotryside A significantly reduced the proliferation of U87 cells in a dose- and time- dependent manner. By flow cytometric analysis, we found that artabotryside A treatment resulted in an increased apoptosis, mitotic arrest, level of ROS and decreased mitochondrial membrane potential in U87 cells in a time-dependent manner. Furthermore, caspase-3 inhibitor, Ac-DEVD-CHO, significantly inhibited the apoptotic effect of artabotryside A, indicating that artabotryside A induced caspase-dependent apoptosis in U87 cells. In addition, artabotryside A is less toxic to normal mouse splenocytes and glial cells. This selective cytotoxicity of artabotryside A against U87 glioma cells as compared to mouse splenocytes and glial cells is advantageous. Thus, artabotryside A might be a safe candidate for prevention and treatment of gliomas. Key words: Flixweed, artabotryside A, anticancer, U87 cell, apoptosis.

Abstract 566: Withaferin A causes autophagy in normal as well as cancerous human breast cells

Abstract Withaferin A (WA), a promising anticancer constituent of traditional Indian (Ayurvedic) medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 (estrogen-independent) and MCF-7 (estrogen-responsive) human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction. We have shown recently that WA-induced apoptosis in human breast cancer cells is initiated by reactive oxygen species (ROS) resulting from inhibition of mitochondrial respiration. Because ROS are frequently implicated in induction of autophagy, which is an evolutionary conserved process for bulk-degradation of macromolecules including organelles, we raised the question of whether WA treatment causes autophagy. In the present study, we have addressed this question using MDA-MB-231 and MCF-7 human breast cancer cells, a spontaneously immortalized and non-tumorigenic normal human mammary epithelial cell line (MCF-10A), and MDA-MB-231 xenografts from control and WA-treated mice. WA treatment resulted in autophagy induction in cancerous (MCF-7 and MDA-MB-231) as well as normal mammary epithelial cells (MCF-10A) as judged by transmission electron microscopy, cleavage of microtubule-associated protein 1 light chain 3 (LC3), formation of acidic vesicular organelles, and punctate pattern of LC3 localization. Immunohistochemical analysis revealed markedly higher expression of LC3 in MDA-MB-231 xenografts from female athymic mice administered i.p. with 4 mg/kg WA compared with control. Autophagy is a protective mechanism against apoptosis by several agents. Interestingly, inhibition of WA-induced autophagy either by a pharmacological agent (3-methyladenine) or siRNA knockdown of critical autophagy regulating proteins ATG5 or Beclin1 did not have any meaningful impact on growth inhibition or apoptotic cell death resulting from WA exposure in MDA-MB-231 or MCF-7 cells. Collectively, these results indicate that WA induces autophagy in breast cancer cells regardless of estrogen receptor expression and this response is not unique to cancerous breast cells. In addition, unlike many other agents, autophagy induction by WA has no influence on its ability to inhibit cell growth or cause apoptosis at least in breast cancer cells. This investigation was supported by US PHS grant CA142604 awarded by the National Cancer Institute. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 566. doi:1538-7445.AM2012-566

Notch2 activation by benzyl isothiocyanate impedes its inhibitory effect on breast cancer cell migration

Benzyl isothiocyanate (BITC) is a promising anticancer constituent of edible cruciferous vegetables with in vivo efficacy against chemically induced as well as oncogene-driven breast cancer in experimental rodents. However, the mechanism underlying anticancer effect of BITC is not fully understood. This study was undertaken to determine the role of Notch signaling in anticancer responses to BITC as this pathway is often hyperactive in human breast cancer. Exposure of MCF-7, MDA-MB-231, and SUM159 human breast cancer cells to pharmacologic concentrations of BITC (2.5 and 5 μM) resulted in cleavage (activation) of Notch1, Notch2, and Notch4, which was accompanied by induction of γ-secretase complex components Presenilin1 and/or Nicastrin. The BITC-mediated cleavage of Notch was associated with its transcriptional activation as revealed by RBP-Jk and Hes-1A/B luciferase reporter assays. Inhibition of cell migration or cell viability resulting from BITC exposure was not influenced by pharmacological suppression of Notch1 using a γ-secretase inhibitor or RNA interference of Notch1 as well as Notch4. On the other hand, the BITC-mediated inhibition of cell migration, but not cell viability, was significantly augmented by siRNA and shRNA knockdown of Notch2 protein. Furthermore, the BITC-mediated inhibition of MDA-MB-231 xenograft growth in vivo was associated with a significant increase in nuclear levels of cleaved Notch2 and Hes-1 proteins. In conclusion, the results of this study indicate that (a) BITC treatment activates Notch2 in cultured and xenografted human breast cancer cells, and (b) Notch2 activation impedes inhibitory effect of BITC on cell migration.

Antiparasitic and Anticancer Constituents of the Endophytic Fungus Aspergillus sp. strain F1544

With the combined goal of finding the best anti-parasitic and anti-cancer activities as well as isolating the bioactive agents and studying their structures and biological properties, we proceeded to perform a small-scale cultivation of Aspergillus sp. strain F1544 using Potato Dextrose, Malt Extract, Czapek Dox and Eight Vegetables media. From the more promising extracts (obtaining using potato dextrose and czapek dox media in large scale) of this fungus, we isolated the five compounds: pseurotin A (1), 14-norpseurotin A (2), FD-838 (3), and pseurotin D (4), and fumoquinone B (5). All compounds showed good antileishmanial and moderate anticancer activities.

Withaferin A-Induced Apoptosis in Human Breast Cancer Cells Is Mediated by Reactive Oxygen Species

Withaferin A (WA), a promising anticancer constituent of Ayurvedic medicinal plant Withania somnifera, inhibits growth of MDA-MB-231 and MCF-7 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo in association with apoptosis induction, but the mechanism of cell death is not fully understood. We now demonstrate, for the first time, that WA-induced apoptosis is mediated by reactive oxygen species (ROS) production due to inhibition of mitochondrial respiration. WA treatment caused ROS production in MDA-MB-231 and MCF-7 cells, but not in a normal human mammary epithelial cell line (HMEC). The HMEC was also resistant to WA-induced apoptosis. WA-mediated ROS production as well as apoptotic histone-associated DNA fragment release into the cytosol was significantly attenuated by ectopic expression of Cu,Zn-superoxide dismutase in both MDA-MB-231 and MCF-7 cells. ROS production resulting from WA exposure was accompanied by inhibition of oxidative phosphorylation and inhibition of complex III activity. Mitochondrial DNA-deficient Rho-0 variants of MDA-MB-231 and MCF-7 cells were resistant to WA-induced ROS production, collapse of mitochondrial membrane potential, and apoptosis compared with respective wild-type cells. WA treatment resulted in activation of Bax and Bak in MDA-MB-231 and MCF-7 cells, and SV40 immortalized embryonic fibroblasts derived from Bax and Bak double knockout mouse were significantly more resistant to WA-induced apoptosis compared with fibroblasts derived from wild-type mouse. In conclusion, the present study provides novel insight into the molecular circuitry of WA-induced apoptosis involving ROS production and activation of Bax/Bak.

Withaferin a suppresses estrogen receptor‐α expression in human breast cancer cells

We have shown previously that withaferin A (WA), a promising anticancer constituent of Ayurvedic medicine plant Withania somnifera, inhibits growth of MCF-7 and MDA-MB-231 human breast cancer cells in culture and MDA-MB-231 xenografts in vivo by causing apoptosis. However, the mechanism of WA-induced apoptosis is not fully understood. The present study was designed to systematically determine the role of tumor suppressor p53 and estrogen receptor-α (ER-α) in proapoptotic response to WA using MCF-7, T47D, and ER-α overexpressing MDA-MB-231 cells as a model. WA treatment resulted in induction as well as increased S15 phosphorylation of p53 in MCF-7 cells, but RNA interference of this tumor suppressor conferred modest protection at best against WA-induced apoptosis. WA-mediated growth inhibition and apoptosis induction in MCF-7 cells were significantly attenuated in the presence of 17β-estradiol (E2). Exposure of MCF-7 cells to WA resulted in a marked decrease in protein levels of ER-α (but not ER-β) and ER-α regulated gene product pS2, and this effect was markedly attenuated in the presence of E2. WA-mediated down-regulation of ER-α protein expression correlated with a decrease in its nuclear level, suppression of its mRNA level, and inhibition of E2-dependent activation of ERE2e1b-luciferase reporter gene. Ectopic expression of ER-α in the MDA-MB-231 cell line conferred partial but statistically significant protection against WA-mediated apoptosis, but not G2/M phase cell cycle arrest. Collectively, these results indicate that WA functions as an anti-estrogen, and the proapoptotic effect of this promising natural product is partially attenuated by p53 knockdown and E2-ER-α.

Bioactivity guided isolation of anticancer constituents from leaves of Alnus sieboldiana (Betulaceae)

The leaves of the Japanese Alnus sieboldiana have been extracted with n-hexane and then with methanol. A bioactivity-guided approach based on MTT assay for growth inhibition and quantitative real-time PCR for TNF-α inhibitory activity was taken to identify the active compounds in EtOAc soluble fraction of the methanol extract. From this active fraction, seven compounds have been isolated and four compounds (pinosylvin, galangin, quercetin and methyl gallate) have been examined for their dose-response effect on the viability of A549 cells and on TNF-α inhibitory activity. Based on MTT assay, all of the four examined compounds inhibit growth of human lung cancer cells. Among four tested compounds only galangin (3,5,7-trihydroxyflavone) significantly inhibited TNF-α gene expression in A549 cells (IC₅₀ = 94 μM). Taken together, this finding suggests that galangin may be useful in cancer prevention.

New aporphine alkaloids and cytotoxic constituents from the root of Illigera luzonensis

Illigera luzonensis Merr. (Hernandiaceae) is a scandent shrub distributed in Philippines, Ryukyus, and southern Taiwan [1]. Aporphine alkaloids, benzylisoquinoline alkaloids, lignans, and their derivatives are widely distributed in plants of the family Hernandiaceae [2], [3]. Many of these compounds exhibit various biological activities, such as cytotoxic, anti-platelet aggregation, vasorelaxing, antioxidant, and antiplasmodial activities [2], [3]. In our studies on the anti-cancer constituents of Formosan plants, many species have been screened for in vitro cytotoxic activity, and I. luzonensis has been found to be an active species. Five new aporphine alkaloids, (S)-N-butyrylcaaverine (1), (S)-N-propionylcaaverine (2), (S)-N-acetylcaaverine (3), (6aR,7R)-N-butyrylnorushinsunine (4), and (6aR,7R,E)-N-(but-2-enoyl)norushinsunine (5), and fifteen known compounds have been isolated and identified from the root of I. luzonensis. Among the isolates, (–)-yatein exhibited cytotoxicities, with IC50 values of 0.81, 0.20, and 0.59µg/mL, respectively, against DLD-1, CCRF-CEM, and HL-60 cell lines. (6aR,7R)-N-Butyrylnorushinsunine (4) exhibited cytotoxicities, with IC50 values of 5.45 and 3.66µg/mL, respectively, against DLD-1 and IMR-32 cell lines. In addition, N-phenethylcinnamamide exhibited cytotoxicities, with IC50 values of 6.84 and 5.79µg/mL, respectively, against DLD-1 and CCRF-CEM cell lines.

Withaferin A inhibits activation of signal transducer and activator of transcription 3 in human breast cancer cells

We have shown previously that withaferin A (WA), a promising anticancer constituent of Ayurvedic medicine plant Withania somnifera, inhibits growth of human breast cancer cells in culture and in vivo in association with apoptosis induction. The present study builds on these observations and demonstrates that WA inhibits constitutive as well as interleukin-6 (IL-6)-inducible activation of signal transducer and activator of transcription 3 (STAT3), which is an oncogenic transcription factor activated in many human malignancies including breast cancer. The WA treatment (2 and 4 μM) decreased constitutive (MDA-MB-231) and/or IL-6-inducible (MDA-MB-231 and MCF-7) phosphorylation of STAT3 (Tyr(705)) and its upstream regulator Janus-activated kinase 2 (JAK2; Tyr(1007/1008)) in MDA-MB-231, which was accompanied by suppression of their protein levels especially at the higher concentration. Exposure of MDA-MB-231 or MCF-7 cells to WA also resulted in suppression of (i) transcriptional activity of STAT3 with or without IL-6 stimulation in both cells; (ii) dimerization of STAT3 (MDA-MB-231) and (iii) nuclear translocation of Tyr(705)-phosphorylated STAT3 in both cells. To our surprise, the IL-6-stimulation, either before or after WA treatment, did not have an appreciable effect on WA-mediated apoptosis in MDA-MB-231 or MCF-7 cell line. The IL-6-stimulated activation of STAT3 conferred a modest protection against WA-mediated suppression of MDA-MB-231 cell invasion. General implication of these findings is that WA can trigger apoptosis and largely inhibit cell migration/invasion of breast cancer cells even after IL-6-induced activation of STAT3, which should be viewed as a therapeutic advantage for this agent.

Abstract A71: Isolation of aglycons and glycosides from Scutellaria baicalensis and exploration of responsible constituents on human breast cancer chemoprevention

Abstract Numerous effective anticancer drugs have been developed from botanical sources. Although the anticancer properties of Scutellaria baicalensis extract (SbE) have been reported, in some of our studies, SbE did not inhibit the growth of breast cancer cells significantly. In certain concentrations, it increased cancer cell growth. To identify the active anticancer constituents in SbE, we isolated a baicalin-knockout fraction (SbF1) and a baicalin-fraction (SbF3). We evaluated the anti-breast cancer activities of the fractions and their constituents on human breast cancer MCF-7 cells. Using a Waters 2960 high performance liquid chromatography (HPLC) instrument and a Phenomenex Prodigy ODS(2) column, we determined the contents in the extract and fractions: scutellarin, baicalin, baicalein and wogonin. The antiproliferative effects were assayed using the modified trichrome stain (MTS) method. The active properties of the fraction/constituents on the cancer cell cycle and on apoptosis were evaluated using a BD FACScan flow cytometer. A one-way ANOVA was used to measure statistical significance. HPLC data showed that the content of scutellarin, baicalin, baicalein and wogonin in SbE were 0.03, 15.67, 5.19 and 1.42%, respectively. The major constituents of SbF1 are the aglycons, baicalein (40.54%) and wogonin (12.35%). SbF3 contains only the glycoside baicalin (16.68%). SbF1 showed a significant antiproliferative effect. Treatment with 100 g/ml of SbF1 for 72 h inhibited MCF-7 cell growth by 81.6% (P<0.01); at the same treatment concentration, SbF3 increased cell growth by 22.6% (P<0.01). Therefore, SbF1 is the active fraction of SbE. Compared to control (48% of G1-phase, 33% of M-phase, and 10% of G2/M-phase), after treatment with 50 g/mL of SbF1 for 72 h, 27% of cells were in G-phase, 41% of cells were in M-phase, and 23% of cells were in G2/M-phase. SbF1 increased the S- and G2/M-phases in MCF-7 cells. After treatment for 48 h, the percentage of early apoptotic cells induced by 50 g/mL of SbF1 was 12.4%; by 100 g/mL, the percentage was 24.2%. SbF1 significantly increased induction of cell apoptosis compared to control, 6.7%. Studying the effects of four flavonoids in SbE, two glycosides (scutellarin and baicalin) and two aglycons (baicalein and wogonin), on MCF-7 cell proliferation showed that baicalein and wogonin significantly inhibited MCF-7 cell growth at 10–100 µM. In contrast, at certain concentrations (5–50 µM), the glycosides scutellarin and baicalin increased cancer cell growth. These phytochemical and biological data suggest that the active anticancer constituents in SbE are flavonoid aglycons. Since the two glycosides promoted MCF-7 cells at certain concentrations, these compounds should be removed from the S. baicalensis extract to ensure chemopreventive activities. Citation Information: Cancer Prev Res 2010;3(1 Suppl):A71.

Terpene Conjugates of the Nigella sativa Seed‐Oil Constituent Thymoquinone with Enhanced Efficacy in Cancer Cells

Thymoquinone (TQ; 1) is a weak anticancer constituent of black seed oil. Derivatives bearing terpene-terminated 6-alkyl residues were tested in cells of human HL-60 leukemia, 518A2 melanoma, multidrug-resistant KB-V1/Vbl cervix, and MCF-7/Topo breast carcinomas, as well as in non-malignant human foreskin fibroblasts. Derivatives with a short four-atom spacer between quinone and cyclic monoterpene moieties were more antiproliferative than analogues with longer spacers. 6-(Menthoxybutyryl)thymoquinone (3a) exhibited single-digit micromolar IC(50) (72 h) values in all four cell lines. It was seven times more active than TQ (1) in 518A2 melanoma cells and four times in KB-V1/Vbl cervix carcinoma cells, while only half as toxic in the fibroblasts. Compound 3a was also not a substrate for the P-gp and BCRP drug transporters of the resistant cancer cells. The caryophyllyl and germacryl conjugates 3e and 3f specifically inhibited the growth of the resistant MCF-7 breast carcinoma cells. Conjugation of TQ with the triterpene betulinic acid via the OH group as in 3g led to a loss in activity, while conjugation via the carboxylic acid afforded compound 4 with nanomolar IC(50) (72 h) activity against HL-60 cells. All anticancer-active derivatives of TQ (1) induced apoptosis associated with DNA laddering, a decrease in mitochondrial membrane potential and a slight increase in reactive oxygen species.

Nuclear matrix protein, prohibitin, was down‐regulated and translocated from nucleus to cytoplasm during the differentiation of osteosarcoma MG‐63 cells induced by ginsenoside Rg1, cinnamic acid, and tanshinone IIA (RCT)

Ginsenoside Rg1, cinnamic acid, and tanshinone IIA (RCT) are effective anticancer and antioxidant constituents of traditional Chinese herbal medicines of Ginseng, Xuanseng, and Danseng. The molecular mechanisms of anticancer effects of those constituents and their targets are unknown. Prohibitin, an inner membrane-bound chaperone in mitochondrion involved in the regulation of cell growth, proliferation, differentiation, aging, and apoptosis, was chosen as a candidate molecular target because of its frequent up-regulation in various cancer cells. We demonstrated that prohibitin existed in the filaments of the nuclear matrix of the MG-63 cell and its expression was down-regulated by the treatment of RCT using proteomic methodologies and Western blot analysis. Immunogold electro-microscopy also found that prohibitin was localized on nuclear matrix intermediate filaments (NM-IF) that had undergone restorational changes after RCT treatment. Prohibitin may function as a molecular chaperone that might interact with multiple oncogenes and tumor suppressor genes. We found that oncogenes c-myc and c-fos and tumor suppressor genes P53 and Rb were regulated by RCT as well and that these gene products co-localized with prohibitin. Our study identified prohibitin as a molecular target of the effective anticancer constituents of Ginseng, Xuanseng, and Danseng that down-regulated prohibitin in nuclear matrix, changed prohibtin trafficking from nucleolus to cytoplasm, and regulated several oncogenes and tumor suppressor genes. Prohibitin downregulation and cellular trafficking from nucleolus to cytoplasm indicated RCT protective roles in cancer prevention and treatment.