Clausenidin Induces Caspase 8-Dependent Apoptosis and Suppresses Production of VEGF in Liver Cancer Cells

Clausena excavata Burm f. is used by traditional healers to treat cancer patients in South East Asia. The use of the plant and its compounds is based on Asian folklore with little or no scientific evidence supporting the therapeutic efficacy The current study aimed to determine the effect of pure clausenidin isolated from C. excavata on caspase-8-induced cell death as well as angiogenesis in the HepG2 hepatocellular carcinoma cell line. Caspase-8 and extrinsic death receptor protein expression was determined using spectrophotometry and protein profile arrays, respectively. Ultrastructural analysis of clausenidin-treated cells was conducted using transmission electron microscopy. In addition, anti-angiogenic effects of clausenidin were investigated by Western blot analysis. Clausenidin significantly (p<0.05) increased the activity of caspase-8 and expression of protein components of the death inducing signaling complex (DISC) in HepG2 cells. Ultrastructural analysis of the clausenidin-treated HepG2 cells revealed morphological abnormalities typical of apoptosis. Furthermore, clausenidin significantly (p<0.05) decreased the expression of vascular endothelial growth factor (VEGF). Therefore, clausenidin is a potential anti-angiogenic agent which may induce apoptosis of hepatocellular carcinoma cells.

Zerumbone Suppresses Angiogenesis in HepG2 Cells through Inhibition of Matrix Metalloproteinase-9, Vascular Endothelial Growth Factor, and Vascular Endothelial Growth Factor Receptor Expressions.

CONTEXT: Due to increase in the number of patients with impaired immunity, the incidence of liver cancer has increased considerably. AIMS: The aim of this study is the investigation the in vitro anticancer effect of zerumbone (ZER) on hepatocellular carcinoma (HCC). MATERIALS AND METHODS: The anticancer mechanism of ZER was determined by the rat aortic ring, human umbilical vein endothelial cells (HUVECs) proliferation, chorioallantoic membrane, cell migration, and proliferation inhibition assays. RESULTS: Our results showed that ZER reduced tube formation by HUVECs effectively inhibits new blood vessel and tissue matrix formation. Western blot analysis revealed that ZER significantly (P < 0.05) decreased expression of molecular effectors of angiogenesis, the matrix metalloproteinase-9, vascular endothelial growth factor (VEGF), and VEGF receptor proteins. We found that ZER inhibited the proliferation and suppressed migration of HepG2 cell in dose-dependent manner. STATISTICAL ANALYSIS USED: Statistical analyses were performed according to the Statistical Package for Social Science (SPSS) version 17.0. The data were expressed as the mean ± standard deviation and analyzed using a one-way analysis of variance. A P < 0.05 was considered statistically significant. CONCLUSION: The study for the first time showed that ZER is an inhibitor angiogenesis, tumor growth, and spread, which is suggested to be the mechanisms for its anti-HCC effect. SUMMARY: Tumor angiogenesis has currently become an important research area for the control of cancer growth and metastasis. The current study determined the effect of zerumbone on factors associated with angiogenesis that occurs in tumor formation. Abbreviations used: ZER: Zerumbone, MMP-9: Matrix metalloproteinase-9, VEGF: Vascular endothelial growth factor, VEGFR: Vascular endothelial growth factor receptor, HUVECs: Human umbilical vein endothelial cells, HCC: Hepatocellular carcinoma, HIFCS: Heat inactivated fetal calf serum, DMSO: Dimethyl sulfoxide, EDTA: Ethyldiaminetetraacetic acid, Ig: Immunoglobulin, CAM: Chorioallantoic membrane, HRP: Horseradish peroxidase, NIH: National Institutes of Health, MTT: Microtetrazolium, SPSS: Statistical Package for Social Science.

Anticancer effect of dentatin and dentatin-hydroxypropyl-ß-cyclodextrin complex on human colon cancer (HT-29) cell line.

INTRODUCTION: Dentatin (DEN) (5-methoxy-2, 2-dimethyl-10-(1, 1-dimethyl-2propenyl) dipyran-2-one), a natural compound present in the roots of Clausena excavata Burm f, possesses pro-apoptotic and antiproliferative effects in various cancer cells. Because of its hydrophobicity, it is believed that its complexation with hydroxy-ß-cyclodextrin (HPßCD) will make it a potent inhibitor of cancer cell growth. In the current work, the molecular mechanisms of apoptosis induced by DEN and DEN-HPßCD complex were demonstrated in human colon HT-29 cancer cells. MATERIALS AND METHODS: After the human colon HT-29 cancer cells were treated with DEN and DEN-HPßCD complex, their effects on the expression of apoptotic-regulated gene markers in mitochondria-mediated apoptotic and death receptor pathways were detected by Western blot analysis and reverse transcription polymerase chain reaction. These markers included caspases-9, 3, and 8, cytochrome c, poly (ADP-ribose) polymerase, p53, p21, cyclin A as well as the Bcl-2 family of proteins. RESULTS: At 3, 6, 12, and 24 µg/mL exposure, DEN and DEN-HPßCD complex significantly affected apoptosis in HT-29 cells through the down-regulation of Bcl-2 and cyclin A in turn, and up-regulation of Bax, p53, p21, cytochrome c at both protein and mRNA levels. DEN and DEN-HPßCD complex also decreased cleaved poly (ADP-ribose) polymerase and induced caspases-3, -8, and -9. CONCLUSION: Results of this study indicate that the apoptotic pathway caused by DEN and DEN-HPßCD complex are mediated by the regulation of caspases and Bcl-2 families in human colon HT-29 cancer cells. The results also suggest that DEN-HPßCD complex may have chemotherapeutic benefits for colon cancer patients.

Correction: Crude Extracts, Flavokawain B and Alpinetin Compounds from the Rhizome of Alpinia mutica Induce Cell Death via UCK2 Enzyme Inhibition and in Turn Reduce 18S rRNA Biosynthesis in HT-29 Cells.

[This corrects the article DOI: 10.1371/journal.pone.0170233.].

Artonin E induces p53-independent G1 cell cycle arrest and apoptosis through ROS-mediated mitochondrial pathway and livin suppression in MCF-7 cells.

Artonin E is a prenylated flavonoid compound isolated from the stem bark of Artocarpus elasticus. This phytochemical has been previously reported to be drug-like with full compliance to Lipinski's rule of five and good physicochemical properties when compared with 95% of orally available drugs. It has also been shown to possess unique medicinal properties that can be utilized in view of alleviating most human disease conditions. In this study, we investigated the cytotoxic mechanism of Artonin E in MCF-7 breast cancer cells, which has so far not been reported. In this context, Artonin E significantly suppressed the breast cancer cell's viability while inducing apoptosis in a dose-dependent manner. This apoptosis induction was caspase dependent, and it is mediated mainly through the intrinsic pathway with the elevation of total reactive oxygen species. Gene and protein expression studies revealed significant upregulation of cytochrome c, Bax, caspases 7 and 9, and p21 in Artonin E-treated MCF-7 cells, while MAPK and cyclin D were downregulated. Livin, a member of the inhibitors of apoptosis, whose upregulation has been noted to precede chemotherapeutic resistance and apoptosis evasion was remarkably repressed. In all, Artonin E stood high as a potential agent in the treatment of breast cancer.

Crude Extracts, Flavokawain B and Alpinetin Compounds from the Rhizome of Alpinia mutica Induce Cell Death via UCK2 Enzyme Inhibition and in Turn Reduce 18S rRNA Biosynthesis in HT-29 Cells.

Uridine-cytidine kinase 2 is an enzyme that is overexpressed in abnormal cell growth and its implication is considered a hallmark of cancer. Due to the selective expression of UCK2 in cancer cells, a selective inhibition of this key enzyme necessitates the discovery of its potential inhibitors for cancer chemotherapy. The present study was carried out to demonstrate the potentials of natural phytochemicals from the rhizome of Alpinia mutica to inhibit UCK2 useful for colorectal cancer. Here, we employed the used of in vitro to investigate the effectiveness of natural UCK2 inhibitors to cause HT-29 cell death. Extracts, flavokawain B, and alpinetin compound from the rhizome of Alpinia mutica was used in the study. The study demonstrated that the expression of UCK2 mRNA were substantially reduced in treated HT-29 cells. In addition, downregulation in expression of 18S ribosomal RNA was also observed in all treated HT-29 cells. This was confirmed by fluorescence imaging to measure the level of expression of 18S ribosomal RNA in live cell images. The study suggests the possibility of MDM2 protein was downregulated and its suppression subsequently activates the expression of p53 during inhibition of UCK2 enzyme. The expression of p53 is directly linked to a blockage of cell cycle progression at G0/G1 phase and upregulates Bax, cytochrome c, and caspase 3 while Bcl2 was deregulated. In this respect, apoptosis induction and DNA fragmentation were observed in treated HT-29 cells. Initial results from in vitro studies have shown the ability of the bioactive compounds of flavokawain B and alpinetin to target UCK2 enzyme specifically, inducing cell cycle arrest and subsequently leading to cancer cell death, possibly through interfering the MDM2-p53 signalling pathway. These phenomena have proven that the bioactive compounds could be useful for future therapeutic use in colon cancer.

Clausenidin from Clausena excavata induces apoptosis in hepG2 cells via the mitochondrial pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Clausena excavata Burm.f. is used locally in folk medicine for the treatment of cancer in South East Asia. AIM OF THE STUDY: To determine the mechanism of action of pure clausenidin crystals in the induction of hepatocellular carcinoma (hepG2) cells apoptosis. MATERIALS AND METHODS: Pure clausenidin was isolated from Clausena excavata Burm.f. and characterized using 1H and 13C NMR spectra. Clausenidin-induced cytotoxicity was determined by MTT assay. The morphology of hepG2 after treatment with clausenidin was determined by fluorescence and Scanning Electron Microscopy. The effect of clausenidin on the apoptotic genes and proteins were determined by real-time qPCR and protein array profiling, respectively. The involvement of the mitochondria in clausenidin-induced apoptosis was investigated using MMP, caspase 3 and 9 assays. RESULTS: Clausenidin induced significant (p<0.05) and dose-dependent apoptosis of hepG2 cells. Cell cycle assay showed that clausenidin induced a G2/M phase arrest, caused mitochondrial membrane depolarization and significantly (p<0.05) increased expression of caspases 3 and 9, which suggest the involvement of the mitochondria in the apoptotic signals. In addition, clausenidin caused decreased expression of the anti-apoptotic protein, Bcl 2 and increased expression of the pro-apoptotic protein, Bax. This finding was confirmed by the downregulation of Bcl-2 gene and upregulation of the Bax gene in the treated hepG2 cells. CONCLUSION: Clausenidin extracted from Clausena excavata Burm.f. is an anti-hepG2 cell compound as shown by its ability to induce apoptosis through the mitochondrial pathway of apoptosis. Clausenidin can potentially be developed into an anticancer compound.

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-ß-Cyclodextrin (DEN-HPßCD).

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-HPßCD (hydroxypropyl-ß-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-HPßCD 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-HPßCD 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-HPßCD 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 HPßCD 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.

Artonin E and Structural Analogs from Artocarpus Species Abrogates Estrogen Receptor Signaling in Breast Cancer.

The increasing rate of mortality ensued from breast cancer has encouraged research into safer and efficient therapy. The human Estrogen receptor α has been implicated in the majority of reported breast cancer cases. Molecular docking employing Glide, Schrodinger suite 2015, was used to study the binding affinities of small molecules from the Artocarpus species after their drug-like properties were ascertained. The structure of the ligand-binding domain of human Estrogen receptor α was retrieved from Protein Data Bank while the structures of compounds were collected from PubChem database. The binding interactions of the studied compounds were reported as well as their glide scores. The best glide scored ligand, was Artonin E with a score of -12.72 Kcal when compared to other studied phytomolecules and it evoked growth inhibition of an estrogen receptor positive breast cancer cells in submicromolar concentration (3.8-6.9 µM) in comparison to a reference standard Tamoxifen (18.9-24.1 µM) within the tested time point (24-72 h). The studied ligands, which had good interactions with the target receptor, were also drug-like when compared with 95% of orally available drugs with the exception of Artoelastin, whose predicted physicochemical properties rendered it less drug-like. The in silico physicochemical properties, docking interactions and growth inhibition of the best glide scorer are indications of the anti-breast cancer relevance of the studied molecules.

In Silico Discovery of Potential Uridine-Cytidine Kinase 2 Inhibitors from the Rhizome of Alpinia mutica.

Uridine-cytidine kinase 2 is implicated in uncontrolled proliferation of abnormal cells and it is a hallmark of cancer, therefore, there is need for effective inhibitors of this key enzyme. In this study, we employed the used of in silico studies to find effective UCK2 inhibitors of natural origin using bioinformatics tools. An in vitro kinase assay was established by measuring the amount of ADP production in the presence of ATP and 5-fluorouridine as a substrate. Molecular docking studies revealed an interesting ligand interaction with the UCK2 protein for both flavokawain B and alpinetin. Both compounds were found to reduce ADP production, possibly by inhibiting UCK2 activity in vitro. In conclusion, we have identified flavokawain B and alpinetin as potential natural UCK2 inhibitors as determined by their interactions with UCK2 protein using in silico molecular docking studies. This can provide information to identify lead candidates for further drug design and development.

Dentatin from Clausena excavata Induces Apoptosis in HepG2 Cells via Mitochondrial Mediated Signaling.

Hepatocellular carcinoma (HCC) is a primary liver cancer with high global incidence and mortality rates. Current candidate drugs to treat HCC remain lacking and those in use possess undesirable side effects. In this investigation, the antiproliferative effects of dentatin (DTN), a natural coumarin, were evaluated on HepG2 cells and DTN's probable preliminary molecular mechanisms in apoptosis induction were further investigated. DTN significantly (p<0.05) suppressed proliferation of HepG2 cells with an IC50 value of 12.0 µg/mL, without affecting human normal liver cells, WRL-68 (IC50>50 µg/mL) causing G0/G1 cell cycle arrest via apoptosis induction. Caspase colorimetric assays showed markedly increased levels of caspase-3 and caspase-9 activities throughout the treatment period. Western blotting of treated HepG2 cells revealed inhibition of NF-κB that triggers the mitochondrial-mediated apoptotic signaling pathway by up-regulating cytoplasmic cytochrome c and Bax, and down-regulating Bcl-2 and Bcl-xL. The current findings suggest DTN has the potential to be developed further as an anticancer compound targeting human HCC.

Phytochemicals from Mangifera pajang Kosterm and their biological activities.

BACKGROUND: Mangifera pajang Kosterm is a plant species from the mango family (Anacardiaceae). The fruits are edible and have been reported to have high antioxidant content. However, the detailed phytochemical studies of the plant have not been reported previously. This study investigates the phytochemicals and biological activities of different parts of Mangifera pajang. METHODS: The plant samples were extracted with solvents of different polarity to obtain the crude extracts. The isolated compounds were characterized using spectroscopic methods. The extracts and isolated compounds were subjected to cytotoxicity tests using human breast cancer (MCF-7), human cervical cancer (HeLa) and human colon cancer (HT-29) cells. The free radical scavenging activity test was conducted using the DPPH assay. Antimicrobial activity tests were carried out by using the disc diffusion method. RESULTS: Phytochemical investigation on the kernel, stem bark and leaves of Mangifera pajang led to the isolation of methyl gallate (1), mixture of benzaldehyde (2) and benzyl alcohol (3), mangiferonic acid (4), 3ß-hydroxy-cycloart-24-ene-26-oic acid (5), 3ß,23-dihydroxy-cycloart-24-ene-26-oic acid (6), lupeol(7) lupenone(8), ß-sitosterol(9), stigmasterol(10), trans-sobrerol(11) and quercitrin (12). Crude ethyl acetate and methanol extracts from the kernel indicated strong cytotoxic activity towards MCF-7 and HeLa cells with IC50 values of less than 10 µg/mL, while petroleum ether, chloroform and ethyl acetate extracts of the stem bark showed strong to moderate activity against MCF-7, HeLa and HT-29 cancer cell lines with IC50 values ranging from 5 to 30 µg/mL. As for the antimicrobial assays, only the ethyl acetate and methanol extracts from the kernel displayed some inhibition against the microbes in the antibacterial assays. The kernel extracts showed highest free radical scavenging activity with IC50 values of less than 10 µg/mL, while the ethyl acetate and methanol extracts of leaves displayed only weak activity in the DPPH assays. CONCLUSIONS: Phytochemical investigations on various parts of Mangifera pajang have identified terpenoids and a flavonol derivative as major constituents. Bioassay studies have indicated that the crude extracts and isolated compounds have potential as naturally-derived anticancer and antimicrobial agents, besides possess high free radical scavenging activity.

Antileukemic effect of zerumbone-loaded nanostructured lipid carrier in WEHI-3B cell-induced murine leukemia model.

Cancer nanotherapy is progressing rapidly with the introduction of many innovative drug delivery systems to replace conventional therapy. Although the antitumor activity of zerumbone (ZER) has been reported, there has been no information available on the effect of ZER-loaded nanostructured lipid carrier (NLC) (ZER-NLC) on murine leukemia cells. In this study, the in vitro and in vivo effects of ZER-NLC on murine leukemia induced with WEHI-3B cells were investigated. The results from 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, Hoechst 33342, Annexin V, cell cycle, and caspase activity assays showed that the growth of leukemia cells in vitro was inhibited by ZER-NLC. In addition, outcomes of histopathology, transmission electron microscopy, and Tdt-mediated dUTP nick-end labeling analyses revealed that the number of leukemia cells in the spleen of BALB/c leukemia mice significantly decreased after 4 weeks of oral treatment with various doses of ZER-NLC. Western blotting and reverse-transcription quantitative polymerase chain reaction assays confirmed the antileukemia effects of ZER-NLC. In conclusion, ZER-NLC was shown to induce a mitochondrial-dependent apoptotic pathway in murine leukemia. Loading of ZER in NLC did not compromise the anticancer effect of the compound, suggesting ZER-NLC as a promising and effective delivery system for treatment of cancers.

Binding mode analysis of zerumbone to key signal proteins in the tumor necrosis factor pathway.

Breast cancer is the second most common cancer among women worldwide. Several signaling pathways have been implicated as causative and progression agents. The tumor necrosis factor (TNF) α protein plays a dual role in promoting and inhibiting cancer depending largely on the pathway initiated by the binding of the protein to its receptor. Zerumbone, an active constituent of Zingiber zerumbet, Smith, is known to act on the tumor necrosis factor pathway upregulating tumour necrosis factor related apoptosis inducing ligand (TRAIL) death receptors and inducing apoptosis in cancer cells. Zerumbone is a sesquiterpene that is able to penetrate into the hydrophobic pockets of proteins to exert its inhibiting activity with several proteins. We found a good binding with the tumor necrosis factor, kinase κB (IKKß) and the Nuclear factor κB (NF-κB) component proteins along the TNF pathway. Our results suggest that zerumbone can exert its apoptotic activities by inhibiting the cytoplasmic proteins. It inhibits the IKKß kinase that activates the NF-κB and also binds to the NF-κB complex in the TNF pathway. Blocking both proteins can lead to inhibition of cell proliferating proteins to be downregulated and possibly ultimate induction of apoptosis.

Phytochemicals from Kaempferia angustifolia Rosc. and their cytotoxic and antimicrobial activities.

Phytochemical investigation on rhizomes of Kaempferia angustifolia has afforded a new abietene diterpene, kaempfolienol (1) along with crotepoxide (2), boesenboxide (3), 2'-hydroxy-4,4',6'-trimethoxychalcone (4), zeylenol (5), 6-methylzeylenol (6), (24S)-24-methyl-5α-lanosta-9(11), 25-dien-3ß-ol (7), sucrose, ß-sitosterol, and its glycoside (8). The structures of the compounds were elucidated on the basis of spectroscopic methods (IR, MS, and NMR). Isolation of 6-methylzeylenol (6), (24S)-24-methyl-5α-lanosta-9(11), 25-dien-3ß-ol (7), and ß-sitosterol-3-O-ß-D-glucopyranoside (8) from this plant species has never been reported previously. The spectroscopic data of (7) is firstly described in this paper. Cytotoxic screening indicated that most of the pure compounds tested showed significant activity with (4) showing the most potent activity against HL-60 (human promyelocytic leukemia) and MCF-7 (human breast cancer) cell lines. However, all extracts and most of the pure compounds tested were found to be inactive against HT-29 (human colon cancer) and HeLa (human cervical cancer) cell lines. Similarly, none of the extracts or compounds showed activity in the antimicrobial testing.

Biomedical properties of a natural dietary plant metabolite, zerumbone, in cancer therapy and chemoprevention trials.

Zerumbone (ZER) is a naturally occurring dietary compound, present in many natural foods consumed today. The compound derived from several plant species of the Zingiberaceae family that has been found to possess multiple biomedical properties, such as antiproliferative, antioxidant, anti-inflammatory, and anticancer activities. However, evidence of efficacy is sparse, pointing to the need for a more systematic review for assessing scientific evidence to support therapeutic claims made for ZER and to identify future research needs. This review provides an updated overview of in vitro and in vivo investigations of ZER, its cancer chemopreventive properties, and mechanisms of action. Therapeutic effects of ZER were found to be scientifically plausible and could be explained partially by in vivo and in vitro pharmacological activities. Much of the research outlined in this paper will serve as a foundation to explain ZER anticancer bioactivity, which will open the door for the development of strategies in the treatment of malignancies using ZER.

Zerumbone-loaded nanostructured lipid carrier induces G2/M cell cycle arrest and apoptosis via mitochondrial pathway in a human lymphoblastic leukemia cell line.

This investigation evaluated the antileukemia properties of a zerumbone (ZER)-loaded nanostructured lipid carrier (NLC) prepared by hot high-pressure homogenization techniques in an acute human lymphoblastic leukemia (Jurkat) cell line in vitro. The apoptogenic effect of the ZER-NLC on Jurkat cells was determined by fluorescent and electron microscopy, Annexin V-fluorescein isothiocyanate, Tdt-mediated dUTP nick-end labeling assay, cell cycle analysis, and caspase activity. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide) assay showed that ZER-NLC did not have adverse effects on normal human peripheral blood mononuclear cells. ZER-NLC arrested the Jurkat cells at G2/M phase with inactivation of cyclin B1 protein. The study also showed that the antiproliferative effect of ZER-NLC on Jurkat cells is through the intrinsic apoptotic pathway via activation of caspase-3 and caspase-9, release of cytochrome c from the mitochondria into the cytosol, and subsequent cleavage of poly (adenosine diphosphate-ribose) polymerase (PARP). These findings show that the ZER-NLC is a potentially useful treatment for acute lymphoblastic leukemia in humans.

Zerumbone-loaded nanostructured lipid carriers: preparation, characterization, and antileukemic effect.

Zerumbone, a natural dietary lipophilic compound with low water solubility (1.296 mg/L at 25°C) was used in this investigation. The zerumbone was loaded into nanostructured lipid carriers using a hot, high-pressure homogenization technique. The physicochemical properties of the zerumbone-loaded nanostructured lipid carriers (ZER-NLC) were determined. The ZER-NLC particles had an average size of 52.68 ± 0.1 nm and a polydispersity index of 0.29 ± 0.004 µm. Transmission electron microscopy showed that the particles were spherical in shape. The zeta potential of the ZER-NLC was -25.03 ± 1.24 mV, entrapment efficiency was 99.03%, and drug loading was 7.92%. In vitro drug release of zerumbone from ZER-NLC was 46.7%, and for a pure zerumbone dispersion was 90.5% over 48 hours, following a zero equation. Using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in human T-cell acute lymphoblastic leukemia (Jurkat) cells, the half maximal inhibitory concentration (IC50) of ZER-NLC was 5.64 ± 0.38 µg/mL, and for free zerumbone was 5.39 ± 0.43 µg/mL after 72 hours of treatment. This study strongly suggests that ZER-NLC have potential as a sustained-release drug carrier system for the treatment of leukemia.

Inclusion Complex of Zerumbone with Hydroxypropyl- ß -Cyclodextrin Induces Apoptosis in Liver Hepatocellular HepG2 Cells via Caspase 8/BID Cleavage Switch and Modulating Bcl2/Bax Ratio.

Zerumbone (ZER) isolated from Zingiber zerumbet was previously encapsulated with hydroxypropyl- ß -cyclodextrin (HP ß CD) to enhance ZER's solubility in water, thus making it highly tolerable in the human body. The anticancer effects of this new ZER-HP ß CD inclusion complex via apoptosis cell death were assessed in this study for the first time in liver hepatocellular cells, HepG2. Apoptosis was ascertained by morphological study, nuclear stain, and sub-G1 cell population accumulation with G2/M arrest. Further investigations showed the release of cytochrome c and loss of mitochondrial membrane potential, proving mitochondrial dysfunction upon the ZER-HP ß CD treatment as well as modulating proapoptotic and anti-apototic Bcl-2 family members. A significant increase in caspase 3/7, caspase 9, and caspase 8 was detected with the depletion of BID cleaved by caspase 8. Collectively, these results prove that a highly soluble inclusion complex of ZER-HP ß CD could be a promising anticancer agent for the treatment of hepatocellular carcinoma in humans.

A Phenylbutenoid Dimer, cis-3-(3',4'-Dimethoxyphenyl)-4-[(E)-3''',4'''-Dimethoxystyryl] Cyclohex-1-ene, Exhibits Apoptogenic Properties in T-Acute Lymphoblastic Leukemia Cells via Induction of p53-Independent Mitochondrial Signalling Pathway.

The current study was designed to evaluate the in vitro cytotoxicity effect of a phenylbutenoid dimer, cis-3-(3',4'-dimethoxyphenyl)-4-[(E)-3 (‴) ,4 (‴) -dimethoxystyryl]cyclohex-1-ene (ZC-B11) isolated from the rhizome of Zingiber cassumunar on various cancer cell line, and normal human blood mononuclear cells, and to further investigate the involvement of apoptosis-related proteins that leads, to the probable pathway in which apoptosis is triggered. Cytotoxicity test using MTT assay showed selective inhibition of ZC-B11 towards T-acute lymphoblastic leukemia cells, CEMss, with an IC50 value of 7.11 ± 0.240 µ g/mL, which did not reveal cytotoxic effects towards normal human blood mononuclear cells (IC50 > 50 µ g/mL). Morphology assessments demonstrated distinctive morphological changes corresponding to a typical apoptosis. ZC-B11 also arrested cell cycle progression at S phase and causes DNA fragmentation in CEMss cells. Decline of mitochondrial membrane potential was also determined qualitatively. In the apoptosis-related protein determination, ZC-B11 was found to significantly upregulate Bax, caspase 3/7, caspase 9, cytochrome c, and SMAC and downregulate Bcl-2, HSP70, and XIAP, but did not affect caspase 8, p53, and BID. These results demonstrated for the first time the apoptogenic property of ZC-B11 on CEMss cell line, leading to the programmed cell death via intrinsic mitochondrial pathway of apoptosis induction.