International Journal of
Molecular Sciences Article
Inducing G2/M Cell Cycle Arrest and Apoptosis through Generation Reactive Oxygen Species (ROS)-Mediated Mitochondria Pathway in HT-29 Cells by Dentatin (DEN) and Dentatin Incorporated in Hydroxypropyl-β-Cyclodextrin (DEN-HPβCD) Al-Abboodi Shakir Ashwaq 1, *, Mothanna Sadiq Al-Qubaisi 1 , Abdullah Rasedee 2 , Ahmad Bustamam Abdul 1,3 , Yun Hin Taufiq-Yap 4 and Swee Keong Yeap 5 1 2 3 4 5
*
MAKNA-UPM, Cancer Research Laboratory, Institute of Bioscience, University Putra Malaysia, 43400 Selangor, Malaysia;
[email protected] (M.S.A.-Q.);
[email protected](A.B.A.) Department of Veterinary Laboratory Diagnosis, Faculty of Veterinary Medicine, University Putra Malaysia, 43400 Selangor, Malaysia;
[email protected] Department of Biomedical Science, Faculty of Medicine & Health Science, University Putra Malaysia, 43400 Selangor, Malaysia Department of Chemistry, Faculty of Science, University Putra Malaysia, 43400 Selangor, Malaysia;
[email protected] LIVES, Institute of Bioscience, University Putra Malaysia, 43400 Selangor, Malaysia;
[email protected] Correspondence:
[email protected]; Tel.: +60-1-121-02-1014
Academic Editor: Anthony Lemarié Received: 5 July 2016; Accepted: 9 September 2016; Published: 18 October 2016
Abstract: Dentatin (DEN), purified from the roots of Clausena excavata Burm f., has poor aqueous solubility that reduces its therapeutic application. The aim of this study was to assess the effects of DEN-HPβCD (hydroxypropyl-β-cyclodextrin) complex as an anticancer agent in HT29 cancer cell line and compare with a crystal DEN in dimethyl sulfoxide (DMSO). The exposure of the cancer cells to DEN or DEN-HPβCD complex leads to cell growth inhibition as determined by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. To analyze the mechanism, in which DEN or DEN-HPβCD complex causes the death in human colon HT29 cancer cells, was evaluated by the enzyme-linked immunosorbent assay (ELIZA)-based assays for caspase-3, 8, 9, and reactive oxygen species (ROS). The findings showed that an anti-proliferative effect of DEN or DEN-HPβCD complex were via cell cycle arrest at the G2/M phase and eventually induced apoptosis through both mitochondrial and extrinsic pathways. The down-regulation of poly(ADP-ribose) polymerase (PARP) which leaded to apoptosis upon treatment, was investigated by Western-blotting. Hence, complexation between DEN and HPβCD did not diminish or eliminate the effective properties of DEN as anticancer agent. Therefore, it would be possible to resolve the conventional and current issues associated with the development and commercialization of antineoplastic agents in the future. Keywords: Dentatin; cytotoxicity; apoptosis; cytostatic; reactive oxygen species (ROS)
1. Introduction Cancer is a devastating disease that wreaks havoc on one’s life. As stated by the World Health Organization (WHO) report, there were 7.9 million deaths in 2007 because of this disease, and it is predicted that this number will grow up to 18 million in 2020 [1]. Specifically, colorectal cancer is the fourth most common malignant disease in the United States [2]. Chronic and metastatic diseases and their treatments continue to be the focus of clinical attention. While there are huge efforts carried Int. J. Mol. Sci. 2016, 17, 1653; doi:10.3390/ijms17101653
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out by most recent researchers to elucidate new molecular target-based molecules, there are also growing interests about natural substances are being considered for application in chemotherapeutic procedures. For instance, phytochemicals, named polyphenols, extracted from different plants parts, such as fruits, leaves, roots, have powerful anticancer effects [3]. Using in vitro cytotoxicity assays for these phytochemical compounds against cancer cells, the mechanisms by which these compounds inhibit cancer growth can be explored. It has been widely revealed that the inhibition mechanism by which these natural compounds work includes inhibition of tumour cell mediated protease activity, promotion of apoptosis and induction of cell cycle arrest [4]. Dentatin (DEN) (5-methoxyl-2,2-dimethyl-10-(1,1-dimethyl-2propenyl) dipyran-2-one), usually purified from Clausena excavata, is one of the vital coumarins which are categorized under polyphenols it is. It has been verified that DEN induces apoptosis in human prostate cancer through inhibition of nuclear factor-κB (NF-κB) nuclear translocation and downregulation of the anti-apoptotic molecules such as Bcl-2, Bcl-xL, and Survivin [5]. In addition, using the mitochondrial signaling pathway, DEN has also stimulated apoptosis in human breast cancer MCF-7 and human hepatocellular carcinoma HepG2 cells lines [6,7]. The drugs’ design and development have two critical factors that affect and limit their therapeutic application, represented by poor aqueous solubility and rate of dissolution [8]. The administration of poorly soluble drugs that belong to class II (high permeability, low solubility) or IV (low permeability, low solubility) of biopharmaceutical classification system (BCS), represents a major challenge [9]. Cyclodextrin (CD) complexation came into view and presented a great interest [10]. In 21st century, using CD represents a novel plan which has emerged to tackle such formulation problems, thus CD has increased the lipophilic compounds’ solubility and their dissolution rate [11]. Cyclodextrins are cyclic molecules composed of six, seven and eight (α, β, and γ-cyclodextrin) glucose units bonded with α-1,4 glycosidic linkages that mold into a truncated cone with a hydrophilic exterior and a hydrophobic cavity [12–14]. The pharmaceutical industry takes great interest in cyclodextrins (CDs) because of their biological nature. Also, the hydrophobic cavities of CDs enable selective binding of different inorganic, organic and biological molecules to form supramolecular complexes without any structural transformations [15–17]. This last point is the most significant property which draws the attention of pharmacologists toward CDs. To overcome the solubility limitations and safety of many compounds associated with innate CDs, several CDs have been synthesized and modified, which include hydroxypropyl and hydroxyethyl CDs [18], carboxymethyl CDs [19], methylated and alkylated cyclodextrins [20], sulfoalkyl ether CDs [21], and branched CDs [14,22]. Hydroxypropyl-β-cyclodextrin (HPβCD) is a characteristic hydroxypropyl CD, which is commonly used as a complex agent to improve the aqueous solubility of many drugs [23,24]. We used HPβCD to formulate an inclusion complex with DEN by freeze-drying technique. The aim of this work was to study the cytotoxicity of both pure DEN dissolved in dimethyl sulfoxide (DMSO) alone and DEN incorporated into HPBCD cavity against human colon cancer HT29 cell line. Moreover, we determine the cell cycle arrest and the death pathway that the treated cells responded to using our compounds by cytometry analysis and quantifications of some apoptotic proteins, respectively. Eventually, it would be fruitful to move the compounds from poor aqueous solubility compounds to compounds dissolved in water without using organic solvent (which has toxic effects on the normal cells). Thereby, these findings will open the door for further investigation of this complex (DEN-HPβCD) via in vivo studies in the future. 2. Results 2.1. Cellular Sensitivity of Cells to DEN and DEN-HPβCD In this study, the MTT assay was used to assess the cytotoxicity activity against the human colon cancer cells (HT29) from both the dissolution of DEN in organic solvent DMSO and DEN incorporation in HPβCD complex. Figure 1A illustrates that DEN exhibited strikingly high cytotoxicity impact
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against human colon cancer cells when used in a dose-dependent manner, and with high concentration, J. Mol. Sci. 2016,especially 17, 1653 20 these Int. impacts were significant (p < 0.05). Consequently, the treated cells exhibited 3aofgradual decline in viability (p < 0.05) in comparison to the untreated cells. The concentration of DEN that causes concentration of DEN that causes death of 50% of tested cells or the IC50 value of DEN was found deathThe of 50% of tested cells or the IC50 value of DEN was found to be 5.6 µg/mL. Earlier research to be 5.6 µg/mL. Earlier research performed by [7] revealed similar toxicity against Estrogen Receptor performed by [7] revealed similar toxicity against Estrogen Receptor positive (ER+) MCF-7 where positive (ER+) MCF-7 where IC50 value was at 6.1 µg/mL. In the study mentioned above, DEN IC50 value was at 6.1 µg/mL. In the study mentioned above, DEN exhibited fewer side-effects on exhibited fewer side-effects on the normal cells in comparison to the cancer cells. The DEN-HPβCD the normal cells comparison to inhibition the cancerofcells. The DEN-HPβCD complex also exhibited growth complex alsoinexhibited growth treated cells with IC50 at 8.5 µg/mL, as shown in Figure inhibition 1B. of treated cells with IC50 at 8.5 µg/mL, as shown in Figure 1B.
Figure 1. (A) Cytotoxicity of DEN (Dentatin) against human colon cancer cells (HT29). The cells were
Figure 1. (A) Cytotoxicity of DEN (Dentatin) against human colon cancer cells (HT29). The cells were plated in 96-well plates and then exposed to 100, 50, 25, 6.25, 3.125 and 1.25 µg/mL of DEN for 72 h. platedThe in viability 96-well of plates and then exposed to 100, 50, 25, 6.25, 3.125 and 1.25 µg/mL of DEN for 72 h. treated cells were measured by using an MTT assay. Mean ± standard deviation (SD). The viability of treated cells measured bywith using an MTTcells; assay. ± standard deviation (SD). (n = 3 well/treatment). * were p < 0.05 compared untreated (B)Mean Cytotoxicity of DEN-HPβCD (n = 3(hydroxypropyl-β-cyclodextrin) well/treatment). * p < 0.05 compared with human untreated cells; (B) cells Cytotoxicity of DEN-HPβCD complex against colon cancer (HT29). The cells were (hydroxypropyl-β-cyclodextrin) complex human colon cells (HT29). The plated in 96-well plates and then exposedagainst to 100, 50, 25, 6.25, 3.125cancer and 1.25 µg/mL of DEN forcells 72 h.were treated cells were measured using an MTT ± standard deviation platedThe in viability 96-well of plates and then exposed to by 100, 50, 25, 6.25,assay. 3.125Mean and 1.25 µg/mL of DEN(SD). for 72 h. (n = 3 well/treatment). * pwere < 0.05measured compared with untreated cells.assay. Mean ± standard deviation (SD). The viability of treated cells by using an MTT (n = 3 well/treatment). * p < 0.05 compared with untreated cells. 2.2. Morphological Examination of Treated Cells
On examining the treated cells under 2.2. Morphological Examination of Treated Cells inverted microscope, it was observed that there were remarkable alterations in the morphology of the cells and significant impacts on the physiology of
On examining theinfluence treated of cells under inverted microscope, it was observed thatexposure there were the cells due to high DEN and DEN-HPβCD. Moreover, with elevated dose and remarkable alterations in the morphology of the cells and significant impacts on the physiology time, this influence was growing. The morphological changes in the treated cells had variousof the cells due to high influence DEN and DEN-HPβCD. with elevatedcells dose andcytoplasmic exposure time, manifestations such as of floating, detached, spherical, Moreover, shrunken, and dispersed with shrinkagewas andgrowing. membraneThe blebbing. However, changes none of these changes in themanifestations untreated this influence morphological in the treatedwere cellsobserved had various cells; the cells exhibited healthy shape and adherence to the basic plates as shown in Figure 2. These and such as floating, detached, spherical, shrunken, and dispersed cells with cytoplasmic shrinkage alterations in morphology and physiology of the cells were accredited to the potential cytotoxicity membrane blebbing. However, none of these changes were observed in the untreated cells; the cells impacts of DEN and its ability to inducetocell death through experiment exhibited healthy shape and adherence the basic platesapoptosis. as shownThe in results Figureof2.this These alterations were similar to the conclusions of earlier research carried out by [6], where increase in the number of in morphology and physiology of the cells were accredited to the potential cytotoxicity impacts of
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DEN and its ability to induce cell death through apoptosis. The results of this experiment were similar toMol. the of Int. J. Sci.conclusions 2016, 17, 1653 of earlier research carried out by [6], where increase in the number 4 of 20 floatingInt.and spherical cells was observed after the cells were treated with DEN in a time-dependent J. Mol. Sci. 2016, 17, 1653 4 of 20 floating and spherical cells was observed afteralso the cells were treated with in a time-dependent manner. Although DEN-HPβCD treated cells exhibited changes inDEN morphology, it was slightly manner. DEN-HPβCD treated in cells also exhibited changes indissolved morphology, it was slightly floatingAlthough and spherical cells wasnoticed observed after thetreated cells were treated with DEN in a in time-dependent less compared to the alterations cells with DEN DMSO (shown in less compared to the alterations noticed in cells treated with DEN dissolved in DMSO (shown in manner. Although DEN-HPβCD treated cells also exhibited changes in morphology, it was slightly Figure 3). Which attributed to accumulated compound in the complex, which then eventually got Figure 3). Which attributed to accumulated compound in the complex, which then eventually got less compared to the alterations noticed in cells treated with DEN dissolved in DMSO (shown in gradually released to the environment. gradually released the environment. Figure 3). Which to attributed to accumulated compound in the complex, which then eventually got gradually released to the environment.
Figure 2. Themorphological morphological changes of HT29 HT29 treated and 6.25 ofofDEN Figure 2. morphological The changes of treated cells with with (3.125 (3.125 andµg/mL) 6.25µg/mL) µg/mL) DEN Figure 2. The changes of HT29 treated cells with (3.125 and 6.25 of DEN dissolved dissolved dimethylsulfoxide sulfoxide(DMSO) (DMSO)for for 24 24 and and 72 72 h. Note: blue dissolved inin dimethyl blue arrows arrowsindicate indicateapoptotic apoptoticcells cells in dimethyl sulfoxide (DMSO) for 24 and 72 h. Note: blue arrows indicate apoptotic cells (200× ). (200×). (200×).
Figure 3. Cont.
Figure 3. 3. Cont. Figure Cont.
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Figure 3. The morphological changes of HT29 treated cells with (3.125 and 6.25 µg/mL) of DEN-
Figure HPβCD 3. The morphological of HT29 treatedapoptotic cells with (3.125 and 6.25 µg/mL) of for 24 and changes 72changes h, Note:ofblue arrows indicate (200×). Figure 3. Thecomplex morphological HT29 treated cells with cells (3.125 and 6.25 µg/mL) of DENDEN-HPβCD complex for 24 and 72 h, Note: blue arrows indicate apoptotic cells (200×). HPβCD complex for 24 and 72 h, Note: blue arrows indicate apoptotic cells (200×). 2.3. Trypan Blue Dye Exclusion The cells incubated for 24 and 72 h at two varying concentrations of 3.125 and 6.25 µg/mL 2.3. Dye 2.3.Trypan TrypanBlue Bluecancer DyeExclusion Exclusion were assessed to evaluate the anti-proliferative activity of DEN and DEN-HPβCD complex. The
The cancer cells for hhatat concentrations ofof 3.125 and trypan blue dyeincubated exclusion technique was72 used totwo analyse the influence of the DEN and DEN-HPβCD The cancer cells incubated for24 24and and 72 twovarying varying concentrations 3.125 and6.25 6.25µg/mL µg/mL were evaluate the the anti-proliferative activity of4, DEN and DEN-HPβCD complex. The trypan complextoon cell proliferation. As depicted in Figure theofHT29 exhibited more sensitivity wereassessed assessed to evaluate anti-proliferative activity DENcells and DEN-HPβCD complex. The towards the anti-proliferative effect of DEN than that of DEN-HPβCD complex. The number of viable blue dye exclusion technique was used to analyse the influence of the DEN and DEN-HPβCD complex trypan blue dye exclusion technique was used to analyse the influence of the DEN and DEN-HPβCD 5 and (2.33 ± 0.3) × treated cells hadAs been decreasedinfrom (7.334,± the 0.3) ×HT29 105 cells/mL to (4.76 ± 0.2) × 10sensitivity on cell proliferation. depicted Figure more towards the complex on cell proliferation. As depicted in Figure 4,cells theexhibited HT29 cells exhibited more sensitivity 105 cells/mL after being treated with 3.125 and 6.25 µg/mL frequently, for 24 h. 72 h of exposure to anti-proliferative effect of DENeffect than that of DEN-HPβCD complex. Thecomplex. number of viable treated cells towards theand anti-proliferative DEN than that of DEN-HPβCD of viable 3.125 6.25 µg/mL of DEN wasofsufficient to decrease viable treated 5cells to (2.63 The ± 0.3)number × 105 and 5 cells/mL 5 cells/mL had been decreased from (7.33 ± 0.3) × 10 to (4.76 ± 0.2) × 10 and (2.33 ± 0.3) × 10 5 5 5 cells/mL. treated(1.56 cells± 0.3) had×been decreased fromwith (7.33 ± 0.3) ×cells, 10 cells/mL to(19.63 (4.76±±0.3) 0.2)× 10 × 10 and (2.33 105 cells/mL compared untreated which were The ± 0.3) × after being with 3.125 6.25 µg/mL for 24 h. 72 for h of24DEN-treated exposure 3.125 and HT29treated cells lowerand sensitivity to DEN-HPβCD complex compare with 105 cells/mL afterexhibited being treated with 3.125 and frequently, 6.25 µg/mL frequently, h. 72 h oftocells. exposure to 5 and 6.25 µg/mL of DEN was sufficient to decrease viable treated cells to (2.63 ± 0.3) × 10 5 After 24 h, the count of viable HT29 cells treated with DEN-HPβCD complex at a concentrations of 3.125 and 6.25 µg/mL of DEN was sufficient to decrease viable treated cells to (2.63 ± 0.3) × 10 and 5 cells/mL. 5 cells/mL µg/mL stood at (6.50with ±with 0.5)untreated ×untreated 105 and (3.cells, 53 ± 0.2) × 105 were cells/mL, while that for untreated (1.56 0.3) 106.25 compared cells, which were(19.63 (19.63 0.3) × 5 cells/mL 5 10 (1.56±± 3.125 0.3) × ×and 10 compared which ±± 0.3) × 10 cells/mL. The 5 cellscells was exhibited at (7.33 ± 0.3) × 10 sensitivity cells/mL. Furthermore, the viable HT29 cells declinedwith steeply from (4.06 cells. The HT29 lower toDEN-HPβCD DEN-HPβCD complex compare DEN-treated HT29 cells exhibited lower sensitivity to complex compare with DEN-treated cells. ± 0.4) × 105 to (1.70 ± 0.09) × 105 when exposed to DEN-HPβCD complex at a concentration of 3.125 After h, the count of viable HT29 cells cells treated treated with withDEN-HPβCD DEN-HPβCDcomplex complexatata aconcentrations concentrations After24 24 h,6.25 theµg/mL countfor of 72 viable HT29 of and h which was significant compared untreated cells, which were (19.63 ± 5 andwith 5 cells/mL, of 3.125 and 6.25 µg/mL stood at (6.50 ± 0.5) × 10 (3. 53 ± 0.2) × 10 while that 5 5 3.125 and 0.3) 6.25 × 105 µg/mL µg/mL. stood at (6.50 ± 0.5) × 10 and (3. 53 ± 0.2) × 10 cells/mL, while that for untreated for untreated cells±was (7.33 ± 0.3) × 105 cells/mL. the viable HT29 cellsfrom declined 5 cells/mL. cells was at (7.33 0.3) at × 10 Furthermore, theFurthermore, viable HT29 cells declined steeply (4.06 5 5 when exposed to DEN-HPβCD complex at steeply from (4.06 ± 0.4) × 10 to (1.70 ± 0.09) × 10 5 5 ± 0.4) × 10 to (1.70 ± 0.09) × 10 when exposed to DEN-HPβCD complex at a concentration of 3.125 aand concentration 3.125 6.25 µg/mL for 72 h which was significant compared untreated cells,± 6.25 µg/mLoffor 72 and h which was significant compared with untreated cells, with which were (19.63 5 which were (19.63 ± 0.3) × 10 µg/mL. 5 µg/mL. 0.3) × 10
Figure 4. Trypan blue exclusion assay for cell viability of HT29 treated cells after 24 and 72 h of treatment with DEN and /or DEN-HPβCD complex. Mean ± standard deviation (SD). (n = 3 well/treatment). * p < 0.05 compared with untreated cells.
2.4. Determination of Apoptosis by Using AO/PI Double Staining Cells treated with DEN and DEN-HPβCD complex were examined under inverted microscope (AxiO4.Vert.A1 FL-LED, Nikon Corporation, Tokyo, Japan),ofand it was observed 2424 h that there Figure Trypan blue exclusion assay forviability cell viability HT29 treated cells after72 and 72 h of Figure 4. Trypan blue exclusion assay for cell of HT29 treated cells after 24after and h of treatment was moderate apoptosis in the form of nuclear chromatin condensation and blebbing in the(SD). cells.(n On= 3 treatment with DEN and /or DEN-HPβCD complex. Mean ± standard deviation with DEN and /or DEN-HPβCD complex. Mean ± standard deviation (SD). (n = 3 well/treatment). the other hand, no noticeable apoptosis was found in the negative control group and the cells were * pwith < 0.05 compared with untreated cells. * well/treatment). p < 0.05 compared untreated cells. seen to have undamaged green nuclei. In addition to this, early-stage apoptotic cells in the form of crescent-shaped or granular yellow-green acridine orange (AO) nuclear staining were observed in 2.4.Determination Determination Apoptosis byUsing Using AO/PI Double Staining 2.4. ofofApoptosis by the experimental group (shown in AO/PI FiguresDouble 5 and Staining 6). Within the cells, there was asymmetrical localization staining. number of early-stage apoptotic cellsexamined increased with an inverted increase inmicroscope the Cells treatedofwith DENThe and DEN-HPβCD complex were under
Cells treated with DEN and DEN-HPβCD complex were examined under inverted microscope (AxiOVert.A1 Vert.A1FL-LED, FL-LED,Nikon NikonCorporation, Corporation,Tokyo, Tokyo,Japan), Japan),and andititwas wasobserved observedafter after24 24hhthat thatthere there (AxiO wasmoderate moderate apoptosis apoptosis in and blebbing in the cells. On was in the the form formof ofnuclear nuclearchromatin chromatincondensation condensation and blebbing in the cells. the other hand, no noticeable apoptosis was found in the negative control group and the cells were On the other hand, no noticeable apoptosis was found in the negative control group and the cells were seento tohave haveundamaged undamagedgreen greennuclei. nuclei.In Inaddition additionto tothis, this,early-stage early-stageapoptotic apoptoticcells cellsin inthe theform formof of seen crescent-shapedororgranular granularyellow-green yellow-green acridine orange (AO) nuclear staining observed in crescent-shaped acridine orange (AO) nuclear staining werewere observed in the the experimental group (shown in Figures 5 and 6). Within the cells, there was asymmetrical localization of staining. The number of early-stage apoptotic cells increased with an increase in the
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experimental group (shown in Figures 5 and 6). Within the cells, there was asymmetrical localization of Int. J. Mol. Sci. 2016, 17, 1653 6 of 20 staining. The number of early-stage apoptotic cells increased with an increase in the concentrations of of DEN and DEN-HPβCD complex. At high concentrations of 12 µg/mL, late-stage DENconcentrations and DEN-HPβCD complex. At high concentrations of 12 µg/mL, late-stage apoptotic cells were apoptotic cells were observed, which had asymmetrically localized and concentrated orange observed, which had asymmetrically localized and concentrated orange nuclear propidiumnuclear iodide (PI) propidium iodide (PI) staining, and which eventually converted to necrosis cells at a concentration staining, and which eventually converted to necrosis cells at a concentration of 24 µg/mL. An uneven of 24 µg/mL. An uneven orange-red fluorescence was observed at the periphery of necrotic cells as orange-red fluorescence was observed at the periphery of necrotic cells as they expanded in volume. they expanded in volume. It appeared that the cells were undergoing the process of disintegration. It appeared that theresults cells were undergoing of disintegration. results The experiment indicated that whenthe theprocess cells were treated with DENThe andexperiment DEN-HPβCD indicated thatinwhen the cells were treatedmorphological with DEN and DEN-HPβCD complex in a dose-dependent complex a dose-dependent manner, changes related to apoptosis were induced in manner, morphological changes related to apoptosis were induced in the cells. the cells.
Figure 5. (A) Fluorescent micrographs of double-stained HT29 cells (acridine orange and propidium
Figure 5. (A) Fluorescent micrographs of double-stained HT29 cells (acridine orange and propidium iodide). Untreated cells appeared normal structure without any features of apoptosis and necrosis. iodide). Untreated cells appeared normal structure without any features of apoptosis and necrosis. The early apoptosis features were seen after treatment with DEN (3 µg/mL) representing intercalated The early apoptosis features werewith seenfragmented after treatment withBlebbing DEN (3after µg/mL) acridine orange (bright green) DNA and 24 h. representing The hallmarksintercalated of late acridine orange (bright green) with fragmented DNA andafter Blebbing 24with h. The hallmarks of late apoptosis which represented by orange colour were noticed 24 h ofafter treated (6 µg/mL) which apoptosis which represented colour were noticed after 24colour h of treated µg/mL) which increased with increasing by theorange concentration. Cells with bright red and bigwith size (6 representing secondary werethe visible after treated Cells with higher concentration at 24 and h. VI, viable BL, increased withnecrosis increasing concentration. with bright red colour big sizecells; representing blebbing of the cell membrane; CC, treated chromatin condensation; LA, late apoptosis; SN,VI, secondary secondary necrosis were visible after with higher concentration at 24 h. viable cells; necrosis; AB, apoptotic bodies. Images representative of one of the experiment; (B) BL, blebbing of the cell membrane; CC, are chromatin condensation; LA,three late similar apoptosis; SN, secondary Count cells on different microphotographs. Note: the magnification is 40×. * p < 0.05 compared with necrosis; AB, apoptotic bodies. Images are representative of one of the three similar experiment; untreated cells. (B) Count cells on different microphotographs. Note: the magnification is 40×. * p < 0.05 compared with untreated cells.
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Figure 6. (A) Fluorescent micrographs of double-stained HT29 cells (acridine orange and propidium
Figure 6. (A) Fluorescent micrographs of double-stained HT29 cells (acridine orange and propidium iodide). Untreated cells appeared normal structure without any features of apoptosis and necrosis. iodide). Untreated cells appeared normal structure without any features of apoptosis and necrosis. The early apoptosis features were seen after treatment with DEN-HPβCD complex (3 µg/mL) The early apoptosis features were seen after treatment with DEN-HPβCD complex (3 µg/mL) representing intercalated acridine orange (bright green) with fragmented DNA and Blebbing after 24 representing intercalated acridine orange (bright green) with fragmented DNA and Blebbing after 24 h. h. The hallmarks of late apoptosis which represented by orange colour were noticed after 24 h of Thetreated hallmarks ofµg/mL) late apoptosis which represented by orange colour were noticed after 24 of treated with (6 which increased with increasing the concentration. Cells with bright redhcolour with (6 µg/mL) which increased with increasing the concentration. Cells with bright red colour and big size representing secondary necrosis were visible after treated with higher concentration at and big24 size secondary necrosis were after treated with higher concentration at 24 h. h. representing VI, viable cells; BL, blebbing of the cellvisible membrane; CC, chromatin condensation; LA, late VI,apoptosis; viable cells; BL, blebbing of the cell membrane; CC, chromatin condensation; LA, late apoptosis; SN, secondary necrosis; AB, apoptotic bodies. Images are representative of one of the three SN,similar secondary necrosis; AB, apoptotic bodies. microphotographs. Images are representative of one of the three experiment; (B) Count cells on different Note: the magnification is 40×.similar *p < 0.05 compared withcells untreated cells. microphotographs. Note: the magnification is 40×. * p < 0.05 experiment; (B) Count on different compared with untreated cells. 2.5. Detection of Caspase-9, 3 and 8 Activity
2.5. Detection of Caspase-9, 3 and 8executive Activity enzymatic marker of apoptosis, while Caspase-9 and 8 are Caspase-3 is a significant initiators for the mitochondria-mediated (intrinsic and extrinsic) apoptotic pathway. By using the Caspase-3 is a significant executive enzymatic marker of apoptosis, while Caspase-9 and 8 are Caspase-3/9 Colorimetric Assay Kit and Caspase-8 IETD-R110 Fluorometric and Colorimetric Assay initiators for the mitochondria-mediated (intrinsic and extrinsic) apoptotic pathway. By using the Kit, the caspase-3, 9 and 8 activities of DEN and DEN-HPβCD complex-treated cells were assessed. Caspase-3/9 Colorimetric Assay Kit and Caspase-8 IETD-R110 Fluorometric and Colorimetric Assay Figure 7 depicts the considerable increase in activity of all caspases in the treated HT29 cells, Kit,compared the caspase-3, activitiescells. of DEN and DEN-HPβCD complex-treated were assessed. to that9ofand the 8untreated The caspase-3, 9 and 8 activities appear to becells almost parallel Figure 7 depicts the considerable increase in activity of all caspases in the treated HT29 cells, compared to the increase in doses of DEN and DEN-HPβCD complex. The HT29 cells that were treated with 12 to that of the untreated cells. The caspase-3, 9 and 8 activities appear to beactivity. almost parallel to the increase µg/mL of DEN or DEN-HPβCD complex exhibited the highest caspases Moreover, after 24 in doses DEN and DEN-HPβCD complex.6.9, The HT29 cells thatmore werefrequently treated with h, the of caspase-3, 9 and 8 activity increased 7 and 1.28 times with12 anµg/mL increaseofinDEN
or DEN-HPβCD complex exhibited the highest caspases activity. Moreover, after 24 h, the caspase-3, 9 and 8 activity increased 6.9, 7 and 1.28 times more frequently with an increase in the dose of DEN
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(12 µg/mL) in the treated cells, compared to that of the untreated cells. On the contrary, the caspases the declined dose of DEN µg/mL)concentration in the treatedofcells, compared to that untreatedofcells. On the activity at the(12 highest 24 µg/mL. After 24 hofofthe incubation the cells treated contrary, the caspases activity declined at the highest concentration of 24 µg/mL. After 24 h of with DEN-HPβCD complex, it was observed that the Caspase-3, 9 and 8 activity increased to about incubation of the cells treated with DEN-HPβCD complex, it was observed that the Caspase-3, 9 and 5.8, 5.2 and 1.14 times than that in the untreated cells (shown in Figure 7), which again declined at 8 activity increased to about 5.8, 5.2 and 1.14 times than that in the untreated cells (shown in Figure a concentration of 24 µg/mL. 7), which again declined at a concentration of 24 µg/mL.
Figure 7. Cont.
Figure 7. Cont.
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Figure 7. Treatment of HT29 cells with DEN and/or DEN-HPβCD complex showed in activation of caspase-3, 9 and 8. Cellscells were with treatedDEN with 3, 6, 12 andDEN-HPβCD 24 µg/mL for 24 complex h, and thenshowed determination of Figure 7. Treatment of HT29 and/or in activation of caspase-3, 9 and 8 activity was done. Mean ± standard deviation (SD). (n = 3 well/treatment). * p < 0.05 caspase-3, 9 and 8. Cells were treated with 3, 6, 12 and 24 µg/mL for 24 h, and then determination of compared with untreated cells. RT-PCR analysis for caspase-3 mRNA expression was stimulated in caspase-3, treated 9 and 8and activity wascells. done. Mean deviation (SD).caspase-3 (n = 3 well/treatment). untreated β-actin used±asstandard control. Part A represents and β actin mRNA * p < 0.05 comparedexpression with untreated cells.treated RT-PCR for caspase-3 expression was stimulated in in DEN-HT29 cells,analysis Part B represents caspase-3mRNA and β actin mRNA expression in DEN-HPβCD-HT29 cells. Theaseffect of DEN DEN-HPβCDcaspase-3 complex onand apoptosis treated and untreated cells.treated β-actin used control. Partand A represents β actin mRNA proteins in the treated HT29 colon cell linecaspase-3 was determined blot assay. expressionregulatory in DEN-HT29 treated cells, Part B cancer represents andby β Western actin mRNA expression Figure 7. Treatment of HT29 cells with DEN and/or DEN-HPβCD complex showed in activation of Detection of poly(ADP-ribose) polymerase (PARP) in DEN-HPβCD-HT29 cells. The effect DEN and for DEN-HPβCD complex onofapoptosis caspase-3, 9 and 8.treated Cells were treated with 3, 6, 12ofand 24 µg/mL 24 h, and then determination caspase-3, 8Cycle activity wasHT29 done. Mean standard deviation (SD). = 3 well/treatment). * p < 0.05blot assay. regulatory proteins in the treated colon cancer cell line was (n determined by Western 2.6. Induction of9 and Cell Arrest by DEN and±DEN-HPβCD Complex with untreated polymerase cells. RT-PCR analysis for caspase-3 mRNA expression was stimulated in Detection compared of poly(ADP-ribose) (PARP). Further analysis of the cell cycle progression in DEN and DEN-HPβCD complex-treated cells
treated and untreated cells. β-actin used as control. Part A represents caspase-3 and β actin mRNA
was expression carried outinthrough flowtreated cytometry. TheBexperiment results indicate thatmRNA DEN and DEN-HPβCD DEN-HT29 cells, Part represents caspase-3 and β actin expression in complex induced cycle in treated cells at theand G2/M phase. Figure 8 shows there was 2.6. Induction of Cell Cyclecell Arrest bythe DEN andHT29 DEN-HPβCD Complex DEN-HPβCD-HT29 treated cells. The effect of DEN DEN-HPβCD complex on that apoptosis a considerable dose-dependent accumulation cells cell in the phase, accounting forblot 6.69% ± 0.03% regulatory proteins in the treated HT29 colonof cancer lineG2/M was determined by Western assay. Further of the cell cycle progression in DEN and DEN-HPβCD complex-treated cells andanalysis 7.17% ± 0.13% for DEN, respectively, after the cells were treated with 6 and 12 µg/mL Detection of poly(ADP-ribose) polymerase (PARP) concentration of DEN 24 h. At the The sameexperiment time, DEN triggered in cells at was carried out through flowforcytometry. resultsa corresponding indicate thatdecrease DEN and DEN-HPβCD G0/G1 phaseofand aCycle considerably higher population of hypodiploid (sub G1/apoptosis). Thus, our 2.6. Induction DEN and DEN-HPβCD Complex complex induced cell Cell cycle inArrest the bytreated HT29 cells at the G2/M phase. Figure 8 shows that results indicate that a blockage occurred at the G2/M phase, causing cell growth inhibition. Further analysisdose-dependent of the cell cycle progression in DEN and DEN-HPβCD complex-treated there wasFurthermore, a considerable accumulation of cells in the G2/M phase,cells accounting the sub G peaks were further corroborated by the corresponding externalization of carriedand out through flow cytometry. The experiment results indicate thatcells DEN were and DEN-HPβCD for 6.69% was ± 0.03% 7.17% ± 0.13% for DEN, respectively, after the treated with 6 and phosphatidylserine, indicating that apoptosis was interceded by cellular mitosis inhibition. In complex induced cell cycle in the treated HT29 cells at the G2/M phase. Figure 8 shows that there was addition, as shown in Figure 9, the DEN-HPβCD complex appeared more similar activity for DEN in 12 µg/mLaconcentration of DEN for 24 h. At the same time, DEN triggered a corresponding decrease considerable dose-dependent accumulation of cells in the G2/M phase, accounting for 6.69% ± 0.03% treated cells by inducing cell cycle arrest in G2/M phase. The ratio of accumulation cells was 7.43% ± in cells at and G0/G1 phase and considerably higher population hypodiploid (sub G1/apoptosis). 7.17% ± 0.13% foraDEN, respectively, after the cells were of treated with 6 and 12 µg/mL 0.2% and 7.62% ± 0.1% after treated with 6 and 12 µg/mL frequently. Furthermore, the sub G peak of DEN fora24blockage h. At the same time, DEN triggered a corresponding decrease in cells atinhibition. Thus, our concentration results indicate that occurred at the G2/M phase, causing cell growth was increased with increasing concentration to be 55.11% ± 0.6% after exposure to 24 µg/mL at 24 h. G0/G1the phase considerably population of hypodiploid G1/apoptosis). externalization Thus, our Furthermore, suband G apeaks were higher further corroborated by the (sub corresponding of results indicate that a blockage occurred at the G2/M phase, causing cell growth inhibition. phosphatidylserine, indicating that apoptosis was interceded by cellular mitosis inhibition. In addition, Furthermore, the sub G peaks were further corroborated by the corresponding externalization of as shown phosphatidylserine, in Figure 9, the DEN-HPβCD complexwas appeared more activity for DENInin treated indicating that apoptosis interceded by similar cellular mitosis inhibition. addition, as shown in arrest Figure 9,inthe DEN-HPβCD appeared more similar activity DEN in ± 0.2% cells by inducing cell cycle G2/M phase.complex The ratio of accumulation cells for was 7.43% cellsafter by inducing cellwith cycle6arrest phase.frequently. The ratio of accumulation cellsthe wassub 7.43% and 7.62%treated ± 0.1% treated andin12G2/M µg/mL Furthermore, G±peak was 0.2% and 7.62% ± 0.1% after treated with 6 and 12 µg/mL frequently. Furthermore, the sub G peak increased with increasing concentration to be 55.11% ± 0.6% after exposure to 24 µg/mL at 24 h. was increased with increasing concentration to be 55.11% ± 0.6% after exposure to 24 µg/mL at 24 h.
Figure 8. Cont.
Figure 8. Cont.
Figure 8. Cont.
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Figure 8. The concentrations (3, 6, 12 and 24 µg/mL) of DEN in HT29-treated cells included G2/M cell Results are represented one the three independent experiments. cells included G2/M cell Figure 8.cycle The arrest. concentrations (3, 6, 12 and 24ofµg/mL) of DEN in HT29-treated Figure 8. The concentrations (3, 6, 12 and 24 µg/mL) of DEN in HT29-treated cells included G2/M cell cycle arrest. Results are represented one of the three independent experiments. cycle arrest. Results are represented one of the three independent experiments.
Figure 9. The concentrations (3, 6, 12 and 24 µg/ml) of DEN-HPβCD complex in HT29-treated cells included G2/M cell cycle arrest. Results are represented one of the three independent experiments.
Figure 9. The concentrations (3, 6, 12 and 24 µg/ml) of DEN-HPβCD complex in HT29-treated cells Figure 9. The concentrations (3, 6, 12 and 24 µg/ml) of DEN-HPβCD complex in HT29-treated cells included G2/M cell cycle arrest. Results are represented one of the three independent experiments. included G2/M cell cycle arrest. Results are represented one of the three independent experiments.
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2.7. Reactive Oxygen Species (ROS) 2.7. Reactive Oxygen Species (ROS)
Dichlorofluorescein fluorescence was used to find intracellular mitochondrial reactive oxygen was used to complex find intracellular mitochondrial reactive species Dichlorofluorescein (ROS) generation influorescence DEN or DEN-HPβCD treated cells. The presence andoxygen growth species (ROS) generation in DEN or DEN-HPβCD complex treated cells. The presence and growth of of the reactive oxygen species (ROS) in the HT29 cells treated with 3, 6, 12 and 24 µg/mL of DEN or the reactive oxygen species (ROS) in the HT29 cells treated with 3, 6, 12 and 24 µg/mL of DEN or DEN-HPβCD complex was considerably greater than that in the untreated cells (shown in Figures 10 DEN-HPβCD complex was considerably greater than that in the untreated cells (shown in Figures 10 and 11). As depicted in Figure 10, with increasing concentration of DEN (3, 6 and 12 µg/mL), there was and 11). As depicted in Figure 10, with increasing concentration of DEN (3, 6 and 12 µg/mL), there a steep increase in ROS (14.25, 46.40, and 63.48), while in the untreated cells, the growth of ROS was was a steep increase in ROS (14.25, 46.40, and 63.48), while in the untreated cells, the growth of ROS significantly slow (4.68). On the other hand, with the highest concentration of DEN (24 µg/mL), was significantly slow (4.68). On the other hand, with the highest concentration of DEN (24 µg/mL), thethe level of ROS which isisless lesscompared compared ROS level in DEN-treated cells level of ROSreduced reducedto to 53.45, 53.45, which to to thethe ROS level in DEN-treated cells (12 (12 µg/mL). µg/mL).Figure Figure1111presented presented that DEN–HPβCD complex induced significant growth of ROS that thethe DEN–HPβCD complex induced significant growth of ROS by by 11.6-fold 11.6-fold (approximately) (approximately)ininHT29 HT29treated treatedcells cellsafter afterthe thecells cellswere wereexposed exposed to 12 µg/mL to 12 µg/mL at at 2424 h, h, compared to untreated cells. However, this increasing was compared to untreated cells. However, this increasing waslow lowcompare comparetotothe theincreasing increasingobserved observedin DEN treated cells.cells. in DEN treated
Figure The concentrations(3,(3,6,6,1212and and24 24µg/mL) µg/mL) of of DEN DEN in Figure 10. 10. The concentrations in HT29-treated HT29-treatedcells cellsinduced inducedROS ROS generation. Results are represented one of the three independent experiments. Note: M1 represented generation. Results are represented one of the three independent experiments. Note: M1 represented cells that producing ROS, M2represented representedthe thecells cellsthat that actively actively producing thethe cells that notnot producing ROS, M2 producingROS. ROS.
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Figure 11. 11. The The concentrations concentrations(3, (3,6,6,12 12and and24 24µg/mL) µg/mL) of of DEN-HPβCD DEN-HPβCD complex in in HT29-treated HT29-treated cells cells Figure inducedROS ROSgeneration. generation.Results Resultsare are represented one of the three independent experiments. Note: induced represented one of the three independent experiments. Note: M1 represented the cells ROS, M2 represented the cells actively producing ROS. M1 represented the that cells not thatproducing not producing ROS, M2 represented thethat cells that actively producing ROS.
2.8. Expression of PARP 2.8. Expression of PARP The expression of PARP in the HT29 cells treated with or without DEN or DEN-HPβCD complex was tested by Western blot analysis. As cells shown in Figure 7, without after 24 DEN h treatment with 3, 6,complex 12 and The expression of PARP in the HT29 treated with or or DEN-HPβCD 24 µg/mL or DEN-HPβCD theinlevels of PARP have decreased was testedofbyDEN Western blot analysis.complex, As shown Figure 7, afterprotein 24 h treatment with 3,significantly 6, 12 and 24 for all treated samples compared with the untreated cells. µg/mL of DEN or DEN-HPβCD complex, the levels of PARP protein have decreased significantly for
all treated samples compared with the untreated cells. 3. Discussion 3. Discussion One of the most interesting area, where the oncologists work recently, is indicating the death pathway that cancer cells behaved following exposure to certain molecule; apoptosis vs. necrosis. One of the most interesting area, where the oncologists work recently, is indicating the death The associated genes with apoptosis, which responded by up-regulating or down-regulating, pathway that cancer cells behaved following exposure to certain molecule; apoptosis vs. necrosis. are commonly screened. Secondary metabolites of many plant have showed anticancer effect against The associated genes with apoptosis, which responded by up-regulating or down-regulating, are cancer cells through apoptosis. Apoptosis can take place directly or indirectly through the modulation commonly screened. Secondary metabolites of many plant have showed anticancer effect against of the course of tumor development via different cellular pathways [25,26]. Recently, the chemotherapy cancer cells through apoptosis. Apoptosis can take place directly or indirectly through the agents for recurrent and metastasis diseases have been developed. But, none of chemotherapy agents is modulation of the course of tumor development via different cellular pathways [25,26]. Recently, the safe even-though they are effective, and able to tackle the disease [27]. In addition, absence of selectivity chemotherapy agents for recurrent and metastasis diseases have been developed. But, none of and short blood circulation time of chemotherapy agents, which in turn, will damage not only cancer chemotherapy agents is safe even-though they are effective, and able to tackle the disease [27]. In cells but also normal and healthy tissue [28]. Thus, there is importunate need to implement novel addition, absence of selectivity and short blood circulation time of chemotherapy agents, which in technology based on combination strategy of cyclodextrin complexation with active compounds to turn, will damage not only cancer cells but also normal and healthy tissue [28]. Thus, there is make them more useful and acceptable therapeutic agents. Furthermore, this combination strategy has importunate need to implement novel technology based on combination strategy of cyclodextrin been provided with some advantages for drug delivery systems such as improvement/enhancement complexation with active compounds to make them more useful and acceptable therapeutic agents. of drug solubility and stability, increasing of its bioavailability and dissolution, reducing of its toxicity, Furthermore, this combination strategy has been provided with some advantages for drug delivery and modifying of its physicochemical characteristics [29]. systems such as improvement/enhancement of drug solubility and stability, increasing of its bioavailability and dissolution, reducing of its toxicity, and modifying of its physicochemical characteristics [29].
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As a result of its chemical properties (like most coumarins), DEN have biomedical applications as anticancer agent [30]. These results focused on the effect of DEN and DEN-HPβCD complex against HT29 cancer cell line. It can be considered as the first documentation on DEN and DEN-HPβCD complex. DEN has induced apoptotic in HT29 cancer cell line through increased ROS, mitochondria-dependent activation of caspase cascade, and G2/M cell cycle arrest. In vitro, cytotoxicity of both DEN and DEN-HPβCD should be screened before testing in animal studies [31,32]. Cytotoxicity determinations include concentration response. This is important because it gives a preclinical evaluation of the range where toxicant-induced cytotoxicity or response takes place [33]. Figure 1A,B showed how HT29 cells responded differently following exposure to DEN and DEN-HPβCD complex. Based on the viability values, it can be said that HT29 cells showed more sensitivity to the DEN dissolved in DMSO than the DEN-HPβCD complex after 72 h of exposure. This difference can be attributed to DMSO’s role and the time needed for drug release. Therefore, exposure time may be prolonged by gradually releasing the DEN from the complex [34]. Moreover, higher resistance was displayed by normal breast MCF10a cells as previously mentioned by [7]. To confirm the selectivity, it can compare the cytotoxicity effects of DEN and DEN-HPβCD complex for cancer cell line with normal cell lines. This is an important part when the evaluation for a newly formulated drug is needed [35]. It was observed that cells treated with DEN and DEN-HPβCD complex exhibited morphological changes closely associated with apoptosis (programmed cell death). Under a light microscope, the morphological characteristics of apoptosis, as a result to treatments with the DEN and DEN-HPβCD complex, are revealed. The alterations referred to early and late phases of apoptotic mechanism in the cells treated with the DEN and DEN-HPβCD complex were demonstrated using the AO/PI double staining fluorescent assay. Under inverted microscope, the dose-dependent increases in apoptotic population were observed. The pathways of apoptosis involve an energy-dependent cascade of molecular events, and are considered to be extremely sophisticated [36]. Apoptosis can take place via two pathways. The first pathway is widely known as the intrinsic or the mitochondrial pathway. The second pathway is the extrinsic or the death-receptor dependent pathway [37]. Free radical generation during cytotoxicity results in the liberation of reactive oxygen species (ROS). Reports reveal that increased ROS levels could cause oxidative damage to cells. ROS is generally localized in the mitochondria and it is important in activating apoptosis and thus inducing cell death [38]. In other words, ROS plays an important role in apoptosis induction by activating the mitochondria intrinsic pathway [7]. Current study revealed that there are increasing in the reactive oxygen species levels in DEN and DEN-HPβCD complex-treated HT-29 cells compared with untreated cells in dose dependent manner. In contrast, the efficacy of the reactive oxygen species decreased when treated cells with highest concentration 24 µg/mL. The results above reveal that DEN and DEN-HPβCD complex are very effective in inducing ROS in treated cells. Hence, the HPβCD didn’t effect on the DEN activity in inducing ROS. An increase in ROS in treated cells was also associated with the regulation of both anti-apoptotic protein (Bcl-2) and glutathione (GSH) [39]. The activation of cysteine proteases, which cleaves numerous specific cellular substrates, greatly affects the intrinsic pathway of apoptosis. An increase in the oxidative stress in the mitochondria resulted into a leakage of cytochrome C from the mitochondrial inter-membrane space to the cytoplasm. This is connected with Apf-1 and caspase 9 and resulted in the formation of an apoptosome complex. Consequently, the executive protein caspase 3 was activated by this apoptosome and cell death was subsequently triggered [39]. Our results revealed that exposure to DEN and DEN-HPβCD complex has significantly increased the levels of caspase 9 and 3 compared with the untreated cancer cells. Based on this result, it can be concluded that apoptosis was induced by the DEN and DEN-HPβCD complex via the intrinsic mitochondria pathway. This result supports previous studies on the role of DEN in up-regulating of caspase 9 and 3 genes in prostate cancer cells [5,6]. Moreover, inducing caspase 8 using DEN and DEN-HPβCD complex in treated cells can indicated the extrinsic pathway of apoptosis is also behaved. Caspase-8 works in two sides; the first one directly activates the downstream effector caspase-3, the second, caspase-8 induces the truncated Bid
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(tBid), which in turn results in the release of cytochrome C from the mitochondria into the cytosol. All these steps result in the activation of caspase-9 and caspase-3 [40]. The highest concentration of DEN (24 µg/mL) also resulted in decreased caspase 9, 8 and 3 levels. This result supports the idea that caspase-mediated apoptosis takes place at low concentrations and, at higher concentrations, treated cells undergo secondary apoptosis associated with proteolysis [41]. With flow cytometry cell cycle analysis revealed that the apoptosis was induced in treated cell after exposure to DEN or DEN-HPβCD complex for (24 h). That can been seen obviously by increasing cells population at the sub-G0 phase. This increasing in Sub-G0 portion was concomitant with the observed increase of G2/M cell ratio, which can also be attributed to the efficacy of the DEN or DEN-HPβCD complex in regulating p53 [42]. That play vital role in sequesters cyclin B1-Cdc2 complex by inducing p21 in response to the DNA damage that took place after treatment [43]. In turn, p53 can induce apoptosis by up-regulating of various of apoptosis-associated genes [42,44]. For further investigation and validation of enzyme-linked immunosorbent assay ELIZA and cytometry findings, western blot assay was done to measure expression levels of PARP protein. The fragmentation of DNA was induced by extrinsic and intrinsic pathway, at this time the cell tries to prevent and repair with the help of poly(ADP-ribose) polymerase (PARP) [45]. The preventing process of DNA fragmentation that the cell attempts to do were inhibited by treatment with DEN or DEN-HPβCD complex that in turn down regulated PARP and making it unable to function properly. Moreover, Molecular mechanism by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis showed increasing in the expression of caspase-3 after exposure to DEN or DEN-HPβCD complex. However, there is poor knowledge about physiological effects of our formulation on human body if applied as real medication. The route of administration and the mechanism of elimination of this carrier are entirely unknown. Because of that, this study offer additional avenues to exploit this complex in vivo models studies for future investigations. 4. Materials and Methods 4.1. Materials For this experiment, Dulbecco’s modified Eagle’s medium (DMEM), 3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), Trypsin, trypan blue dye, phosphate-buffered saline (PBS), and were purchased from Sigma Chemical Company (St. Louis, MO, USA). The HPβCD (purity ≥ 98%) used in this investigation was procured from Sigma Aldrich (Taufkirchen, Germany). All the chemical materials and reagents and used were analytical grade, and ultrapure water was used during all the experimental steps. 4.2. Cell Culture Human colon cancer HT29 cells bought from ATCC, Rockville, MD, USA and grown in Dulbecco’s Modified Eagles medium (DMEM) enhanced with 1% amphotericin B, 1% penicillin-streptomycin and 10% fetal bovine serum to grow the cells. A humidified incubator at a temperature of 37 ◦ C and with 5% CO2 was used to incubate the flasks that contained cancer cells. 4.3. Preparation of the Inclusion Complex Freeze-drying technique was utilized to prepare the DEN-HPβCD complex. This required preparation of 1:1 molar ratio DEN solutions in HPβCD 0.3264 g DEN was dissolved in 5 mL chloroform and mixed with 1.4 g HPβCD that was diluted in 20 mL of ultra-pure water. After stirring the mixture for 72 h at room temperature, the mixture was filtered using 0.45 m filter paper. The filtered clear solution was frozen at −80 ◦ C, and then freeze-dried for 24 h at −55 ◦ C.
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4.4. Cytotoxicity MTT Assay In a 96-well tissue culture plate, the HT29 cells line were plated at 2 × 103 cells/well by adding 200 µL of a 1 × 104 cells/mL suspension to each well. To ensure attachment at 30% to 40% confluency, the plates were incubated for sufficient amount of time. The already present media was removed and fresh media (200 µL) was added containing DEN or DEN-HPβCD complex of different concentrations (1.56–100 µg/ mL), while the last row was left untreated. For 72 h, the plates were incubated at a temperature of 37 ◦ C and 5% of CO2 . On completion of the incubation with the compounds, the media was removed and the cells were washed three times with PBS buffer so that the cells did not have any trace of DEN or DEN-HPβCD complex, and then a fresh media was added. To every well, the MTT solution (20 µL) with a total volume of 200 µL was added and mixed gradually with the media, and later incubated at 37 ◦ C with 5% CO2 for 4 to 6 h. Subsequently, the MTT-containing medium was aspirated off and gently replaced with DMSO (200 µL per well) so that the formazin crystals could be dissolved. The plates were read at 570 nm in a microtiter plate reader. Using the dose-response curves of each compound, the concentration of drug required to stop the cell growth by 50% (IC50 ) was determined. 4.5. Microscopic Examination of Cell Morphology In the six-well plates, the HT29 cells (1 × 105 cells/well) were seeded and incubated overnight so that the cells hold on to the plates. After this, the cells were exposed to 3.125 and 6.25 µg/mL of DEN and DEN-HPβCD complex for 24 and 72 h, respectively. A light-inverted microscope was then used to observe the changes in membrane and normal morphology of the cells. 4.6. Trypan Blue Exclusion Assay To determine the antiproliferative effect of DEN or DEN-HPβCD complex, at first, the HT29 cells were seeded (1 × 105 cells/well in DMEM) in the six-well tissue culture plates. After 24 h of incubation that enabled the cells to attach to the plates, the cells undergoing exponential growth were exposed to DEN or DEN-HPβCD complex at concentrations of 3.125 and 6.25 µg/mL. Then, for 24 to 72 h, the plates were incubated at a temperature of 37 ◦ C, and CO2 presence of 5%. On completion of the incubation, the medium was removed, and the plates were cleaned with cold phosphate-buffered saline solution to remove the dead cells and replenished with a fresh medium of 1 mL of 0.05% (2 mg/mL) trypsin-ethylene diamine tetra acetic acid. Then, for 10 to 15 min, the plates were incubated at a temperature of 37 ◦ C, till most of the cells confirmed as microscopically detached. The cells were then collected through centrifugation of the cell suspension at 1000 rpm for 10 min and discarding the supernatant. The resultant cell suspension (20 µL) was then blended with 20 µL of 0.4% trypan blue solution which led to the resuspension of the cells. Thereafter, by using a hemocytometer chamber, the dye-excluding viable cells were microscopically counted. 4.7. Morphological Evaluation of Apoptotic Cells by Acridine Orange (AO) Propidium Iodide (PI) Double Staining To further confirmation role of DEN and DEN-HPβCD complex in processing cells death, acridine orange (AO) and propidium iodide (PI) double-staining were used, where the cells were examined under the (inverted microscope AxiO Vert.A1 FL-LED, Nikon Corporation, Tokyo, Japan). The treatment was carried out in the six-well tissue culture plates. The plating of the cells was done at (1 × 105 cells/well in DMEM) and then the plates were treated with varying concentrations of DEN and DEN-HPβCD complex (3, 6, 12, and 24 µg/mL) in a dose-dependent manner for 24 h. The cells then underwent centrifugation at 300g for 10 min. The supernatant of the cell suspension was discarded and the cells were washed twice with PBS, so that the remaining media could be removed. Ten microliters of fluorescent dyes containing AO (10 mg/mL) and PI (10 mg/mL) in equal volumes were supplemented into the cellular pellet, and the freshly stained cell suspension was released into
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a glass slide and shielded with a cover slip. Under (inverted microscope AxiO Vert.A1 FL-LED), the slides were examined for 30 min till the fluorescence eventually faded. When AO and PI have characteristic to bind with DNA. Out of PI and AO, only AO has the potential to cross the plasma membrane of early and sustainable apoptotic cells. The identification criteria include: (i) viable or sustainable cells that show the presence of green nuclei with undamaged structure; (ii) a bright-green nucleus exhibiting chromatin condensation in the nucleus that reveal early apoptosis; (iii) dense orange spaces of chromatin condensation in the nucleus that exhibit late apoptosis and (iv) undamaged orange nucleus indicating secondary necrosis [5]. 4.8. Caspase-3 and -9 Activity By using the Caspase-3/9 Colorimetric Assay Kit (GenScript, Piscataway Township, NJ, USA) on the HT29 cells that were treated with varying concentrations of DEN or DEN-HPβCD complex (3, 6, 12, and 24 µg/mL) in a dose-dependent manner for 24 h, the extent of caspase-3, 9 activation in the cells was evaluated and measured. The cells were plated at a density of 1 × 106 cell/well as per the manufacturer’s protocol (GenScript). The plated cells were treated with DEN or DEN-HPβCD, and the cell suspension was centrifuged. The cells were then collected and washed with PBS, lysed in 50 µL of cold lysis buffer and left on ice with vortex for 1 h. Thereafter, 50 µL of supernatant of each sample was mixed with 50 µL of 2× reaction buffer and 5 µL (200 µM) of DEVD-pNA substrate (caspase-3)/LEHD-pNA substrate (caspase-9), and the solution was incubated away from light for 4 h at a temperature of 37 ◦ C. Then, the plates were studied at 405 nm. 4.9. Caspase-8 Activity By using the Caspase-8 IETD-R110 Fluorometric and Colorimetric Assay Kit (Biotium, Fremont, CA, USA) on the HT29 cells that were treated with different concentrations of DEN or DEN-HPβCD (3, 6, 12, and 24 µg/mL) in a dose-dependent manner for 24 h, the extent of caspase-8 activation in the cells was evaluated and measured. The plating of the cells was done at a density of 1 × 106 cells/well. Once the cells were treated with DEN or DEN-HPβCD complex (3, 6, 12, and 24 µg/mL) in a dose-dependent manner for 24 h, the cells were collected through the process of centrifugation. The harvested cells or pellets were washed with PBS, lysed in 50 µL of chilled cell lyses buffer, and set down on ice for 10 min. At a temperature of 4 ◦ C, the lysate was centrifuged for 5 min. Then, 50 µL of Assay Buffer was added to the centrifuged supernatant for every sample and blended well, and 5 µL of 1 mM Enzyme Substrate was further added to the samples. The samples were then made to undergo incubation at a temperature of 37 ◦ C for 30 to 60 min. At 520 nm emission and 470 nm excitation, the plates were then readed. 4.10. Cell Cycle Analysis With the use of flow cytometry analysis, the proportion of HT29 cancer cells in the different phases was assessed. The HT29 cells were seeded in the six-well plates at a density of (1 × 106 cells/well) for 24 h, in accordance to the protocol defined by the manufacturer (BD Cycletest plus DNA Kit, BD Biosciences, San Jose, CA, USA). Then the cells were treated with 3, 6, 12 and 24 µg/mL of DEN or DEN-HPβCD complex, and incubated for 24 h. After harvesting the cells, 1 mL of buffer solution was added to the cells to create a resuspension, which was then centrifuged. Each tube was filled with 250 µL of solution a (trypsin buffer) and incubated at room temperature for 10 min. The tubed were then supplemented with 200 µL of solution B (trypsin inhibitor and RNase buffer) and incubated at room temperature for 10 min. 200 µL of cold solution C (PI stain solution) was further added to every tube and incubated using ice for 10 min in the dark. Then using the flow cytometry analysis (FACS Calibur), the cells were studied under 488 nm excitation, while the PI fluorescence was measured under 620–640 nm excitation (Becton-Dickinson, Franklin Lakes, NJ, USA).
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4.11. Measurement of Reactive Oxygen Species (ROS) Level By utilizing the total reactive oxygen species (ROS) assay kit (Ebioscience, San Diego, CA, USA). The production of intracellular reactive oxygen species (ROS) was ascertained. The experiment was carried out by following the instructions of the manufacturer. In six-well plates, the cells were seeded at (1 × 106 cells/well) and then incubated for 24 h. After incubation, the cells were treated with varying concentrations of DEN or DEN-HPβCD complex for 24 h. Then, by using trypsin, the cells were detached. The detached cells were washed with PBS. Thereafter, the cells were treated with 100 µL ROS assay stain solution and incubated for 60 min at a temperature of 37 ◦ C and CO2 presence of 5%. The BD face flow cytometer was then used for further analysis. 4.12. Western Blotting Analysis To determine protein expression, HT29 cells were plated at 1 × 106 cells/well in 6-well plate. Then the cells were treated with different concentrations of DEN or DEN-HPβCD complex for 24 h. Cells were collected and lysed in lysis buffer (50 mM Tris–HCl pH 8.0, 120 mM NaCl, 0.5% NP-40, 1 mM phenylmethylsulfonyl fluoride (PMSF). 40 µg of the protein was resolved on 10% sodium dodecyl sulfate SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Darmstadt, Germany). The membranes were blocked with 5% nonfat dry milk in TBS-T (0.05% Tween 20) for 1 h at room temperature. Membranes were incubated with appropriate primary mouse antibody overnight at 4 ◦ C, followed by incubated with horseradish peroxidase (HRP)-conjugated secondary anti-mouse antibody for 1 h at room temperature. Protein-antibody complexes were detected with chemiluminescence (ECL System) and exposed by autoradiography (ChemiDoc MP imaging System/Bio-Rad, USA) 4.13. RNA Isolation and Reverse Transcription-Polymerase Chain Reaction (RT-PCR) Analysis After treated the cells with different concentrations of DEN or DEN-HPβCD, Total RNA was isolated by using a Trizol RNA isolation kit (Invitrogen, Carlsbad, CA, USA) and stored at −80 ◦ C for subsequent analysis. First-strand cDNA was synthesized from 600 ng of RNA by using iScript cDNA Synthesis kit (Bio-Rad). MyTaq Mix kit (Bioline, Taunton, MA, USA) was used for amplification. The sequences of primers used in RT-PCR were shown in Table 1. The PCR cycling conditions for caspase-3 were 95 ◦ C for 1 min and the amplification was followed by 30 cycles of denaturing at 95 ◦ C for 15 s, annealing at 60 ◦ C for 15 s, and primer extension a 72 ◦ C for 10 s with a final extension at 72 ◦ C for 7 min. The PCR cycling conditions for β-actin were 95 ◦ C for 1min and the amplification was followed by 30 cycles of denaturing at 95 ◦ C for 15 s, annealing at 61 ◦ C for 15 s, and primer extension a 72 ◦ C for 10 s with a final extension at 72 ◦ C for 7 min. The PCR product was analysed on 1.5% agarose gels and DNA bands were visualized by 5× loading buffer and Bio rad gel documentation system/Bio-Rad. All experiments were performed in triplicate. Table 1. The sequences of primers used in RT-PCR. Primers
Forward primer (50 –30 )
Reverse primer (50 –30 )
capspase-3 β-actin
TCA CAG CAA AAG GAG CAG TT TCA CCC TGA AGT ACC CCA TC
CGT CAA AGG AAA AGG ACT CA CCA TCT CTT GCT GCA AGT CC
4.14. Statistical Analysis All the experiments were concluded in triplicate, and the results were reported in terms of mean ± SD. According to the SPSS, statistical analysis was accomplished through the experiments. The analysis of variance was carried out using the ANOVA technique, and a value of p < 0.05 was deemed to be of statistical significance.
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5. Conclusions In conclusion, this study was able to establish that DEN-HPβCD complex is a potent compound that is capable of inducing apoptosis in metastatic colon cancer in vitro. Apoptosis was achieved via multiple cancer-signaling pathways such as Fas mediated, intrinsic, ROS and cell cycle arrest. Further studies are needed to extrapolate the in vitro cytotoxic effects of the complex to in vivo anticancer application. More research is also needed to determine the half-life, stability, and the biologically significant concentrations needed to induce the apoptotic pathway in vivo. Therefore, our findings strongly suggest that the DEN-HPβCD complex deserves further investigation as a potential cancer chemotherapeutic agent. Acknowledgments: This research was supported by science fund research grant (02-01-04-sf1210), Ministry of science, Technology and innovation, Malaysia. The author (Al-Abboodi Shakir Ashwaq) is grateful to University of AL-Qadisiyah, Ministry of Higher Education and Scientific Research, Iraq. Author Contributions: Al-Abboodi Sh Ashwaq, Mothanna Sadiq Al-Qubaisi, Rasedee Abdullah and Ahmad Bustamam Abdul conceived and designed the research, Yun H Taufiq-Yap prepared the DEN-HPβCD complex and provided the facilities for the complex characterization, Swee Keong Yeap performed all analysis involving flow cytometry. Ashwaq wrote the paper. All the authors proofread and gave consent before submission of the manuscript. Conflicts of Interest: The authors declare no conflict of interest.
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