Two new compounds with anticancer and antibacterial activities purified from Fusarium oxysporum LZC03

Antioxidant-guided fractionation of Mammea americana L. seeds resulted in the identification of three new isoprenylated coumarins, mammea B/BA hydroxycyclo F (1), mammea E/BC (2), and mammea E/BD (3). In addition, twelve known isoprenylated coumarins, mammea A/AA (4), mammea A/AA cyclo D (5), mammea A/AA cyclo F (6), mammea A/AC cyclo D (7), mammea A/AD cyclo D (8), mammea B/BA (9), mammea B/BA cyclo F (10), mammea B/BB (11), mammea B/BC (12), mammea B/BD (13), mammea E/BA (14), and mammea E/BB (15), as well as two known flavanols, (+)-catechin (16) and (-)-epicatechin (17) were identified. The fifteen isoprenylated coumarins were screened for their cytotoxicity in the SW-480, HT-29, and HCT-116 human colon cancer cell lines and antioxidant capacities in the DPPH (1,1-diphenyl-2-picrylhydrazyl) free-radical assay. Compounds 1 - 15 exhibited significant cytotoxic activities in the SW-480, HT-29, and HCT-116 human colon cancer cell lines (IC50 ranges 13.9 - 88.1, 11.2 - 85.3, and 10.7 - 76.7 microM, in the three cell lines, respectively) at concentrations comparable to 5-fluorouracil (IC50 = 53.0, 46.1, and 45.1 microM), a drug frequently used for human colon cancer treatment. Compounds 2 - 4, 9, and 11 - 15 displayed high antioxidant activity in the DPPH assay (IC50 range 86 - 135 microM), compounds 1, 5 - 8, and 10, however, had no antioxidant activity (IC50 > 200 microg/mL) in the DPPH assay. The results of these assays were used to study the structure-activity relationships for this class of compounds. In the SW-480 cell line, the three new coumarins, 1 - 3, also exhibited dose-dependent increases in sub-diploid cells by flow cytometry, indicating that they induce apoptosis.

The stilbene derivative, cis-3, 4’, 5-trimethoxy-3’-aminostilbene (stilbene 5c), is a potentially potent antitumor agent that acts via binding to the colchicine-binding pocket in microtubules. Earlier studies have shown that stilbene 5c induces cell death in ovarian cancer cells and leukemic cells (Cao TM et al. Am J Hematol. 2008;83:390-7; Durrant D et al. Gynecol Oncol. 2008; 110:110-7).). The present study was designed to investigate the effectiveness of this microtubule poison against the HCT-116 human colon cancer cell line as well as B16F10 melanoma cells and its mechanisms of action. Time course studies demonstrated that stilbene 5c produces a decrease in cell viability in both cell lines. The capacity of the cells to proliferate was not restored upon removal of the drug after 5 days of exposure. Consistent with the results of the time course studies, β-galactosidase staining indicated that treatment with stilbene 5c also promotes senescence. In addition to senescence, HCT-116 cells and B16F10 melanoma cells treated with stilbene 5c displayed formation of autophagic vesicles by acridine orange staining, which was supported by fluorescence-activated cell sorting (FACS). Further evidence of autophagy was derived from treatment of HCT116 cells expressing an RFP-LC3 construct with stilbene 5c, in which LC3 puncta formation increased in a time-dependent manner. DAPI staining, TUNEL, and Annexin 5 staining indicated that apoptosis is also occurring in stilbene 5c-treated HCT-116 cells. Cell cycle analysis of HCT116 cells demonstrated growth arrest at both G1 and G2/M, and an increase in the subG1 population at days 3 and 5, which correspond to senescence and apoptosis respectively. Interestingly, DAPI and Hoechst staining of HCT116 cells revealed morphological changes in the cell nuclei (binucleated and micronucleated cells), which suggest that mitotic catastrophe may also serve as a mode of cell death after treatment with stilbene 5c. Our studies also indicated that stilbene 5c works in a p53-independent manner. p53-null HCT116 cells demonstrated similar sensitivity to stilbene as p53-wild type HCT116 cells. Consistent with previous studies in other experimental cancer models, this work indicates that stilbene 5c could potentially be effective against melanoma and colon cancer through the promotion of multiple modes of cell death. Citation Format: Moureq R. Alotaibi. Cell death and growth arrest pathways mediating the actions of stilbene 5c in HCT-116 colon cancer cells and B16F10 melanoma cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1658. doi:10.1158/1538-7445.AM2013-1658

The present study demonstrated the anti-tumor effects of the quinoline derivative [5-(3-chloro-oxo-4-phenyl-cyclobutyl)-quinoli-8-yl-oxy] acetic acid hydrazide (CQAH) against colorectal carcinoma. Substantial apoptotic effects of CQAH on HCT116 and LoVo human colon cancer cell lines were observed. Apoptosis was identified based on cell morphological characteristics, including cell shrinkage and chromatin condensation as well as AnnexinV/propidium iodide double staining followed by flow cytometric analysis and detection of apoptosis-associated proteins by western blot analysis. CQAH induced caspase-3 and PARP cleavage, reduced the expression of the anti-apoptotic proteins myeloid cell leukemia-1 and B-cell lymphoma (Bcl) extra large protein and elevated the expression of the pro-apoptotic protein Bcl-2 homologous antagonist killer. In addition, pharmacological inhibition of c-Jun N-terminal kinase (JNK), but not extracellular signal-regulated kinase or p38, significantly reduced CQAH-mediated cell death as well as cleavage of caspase-3 and PARP. Co-treatment of CQAH with the commercial chemotherapeutics 5-fluorouracil and camptothecin-11 significantly improved their efficacies. Comparison of the apoptotic effects of CQAH with those of two illustrated structure-activity associations for this compound type, indicating that substitution at position-4 of the azetidine phenyl ring is pivotal for inducing apoptosis. In conclusion, the results of the present study indicated CQAH and its analogues are potent candidate drugs for the treatment of colon carcinoma.

The antitumor, antibacterial and antioxidant activity, DNA interaction and cathepsin B inhibition of cyclo-ortho-palladated and -platinated compounds [Pd(C,N)]2(μ-X)2 [X=OAc (1), X=Cl (2)] and trans-N,P-[M(C,N)X(PPh3)] [M=Pd, X=OAc (3), M=Pd, X=Cl (4), M=Pt, X=Cl (5)] are discussed [(C,N)=cyclo-ortho-metallated benzophenone imine]. The cytotoxicity of compound 5 has been evaluated towards human breast (MDA-MB-231 and MCF-7) and colon (HCT-116) cancer cell lines and that of compounds 1–4 towards the HCT-116 human colon cancer cell line. These cytotoxicities have been compared with those previously reported for compounds 1–4 towards MDA-MB-231 and MCF-7 cancer cell lines. Compound 3 and 4 were approximately four times more active than cisplatin against the MDA-MB-231 and MCF-7 cancer cell lines, and compound 5, was approximately four times more potent than cisplatin against the HCT-116 cancer cell line. The antibacterial activity of compounds 1–5 was in between the ranges of activity of the commercial antibiotic compounds cefixime and roxithromycin. Complexes 1–2 and 4–5 presented also antioxidant activity. Compounds 1–5 alter the DNA tertiary structure in a similar way to cisplatin, but at higher concentration, and do not present a high efficiency as cathepsin B inhibitors. Compound 5 has not been previously described, and its preparation, characterization, and X-ray crystal structure are reported.

The stilbene derivative, cis-3, 4′, 5-trimethoxy-3′-aminostilbene (stilbene 5c), is a potentially potent antitumor agent that acts via binding to the colchicine-binding pocket in microtubules. Earlier studies have shown that stilbene 5c induces cell death in ovarian cancer cells and leukemic cells (Lee RM et al, 2008; Lee RM et al, 2008). The present study was designed to investigate the effectiveness of this microtubule poison against the HCT-116 human colon cancer cell line and its mechanisms of action. Time course studies demonstrated that stilbene 5c produces a biphasic decrease in cell viability. The capacity of the cells to proliferate was not restored upon removal of the drug after 6 days of exposure. Consistent with the results of the time course studies, β-galactosidase staining indicated that treatment with stilbene 5c also promotes senescence. In addition to senescence, stilbene 5c-treated HCT-116 cells displayed formation of autophagic vesicles by acridine orange staining, which was supported by fluorescence-activated cell sorting (FACS). Further evidence of autophagy was derived from treatment of HCT116 cells carrying an RFP-LC3 construct with stilbene 5c, in which LC3 puncta formation increased in a time-dependent manner. DAPI staining, TUNEL, and Annexin 5 staining indicated that apoptosis is also occurring in stilbene 5c-treated HCT-116 cells. Cell cycle analysis demonstrated growth arrest at both G1 and G2/M, and an increase in the subG1 population at days 3 and 5, which correspond to senescence and apoptosis respectively. Interestingly, DAPI and Hoechst staining revealed morphological changes in the cell nuclei (binucleated and micronucleated cells), which suggest that mitotic catastrophe may also serve as a mode of cell death after treatment with stilbene 5c. Consistent with previous studies in other experimental cancer models, this work indicates that stilbene 5c could potentially be effective against colon cancer through the promotion of multiple modes of cell death. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3957. doi:1538-7445.AM2012-3957

Colorectal cancer is the third most frequently occurring cancer in both men and women and is the second leading cause of cancer deaths in the United States. Despite advances in the treatment of this cancer, the five-year averaged survival rate for all stages has only increased to 66% and more than 49,000 deaths of colon cancer in the United States in 2011. Hence, novel therapeutic approaches of more effective treatments are much needed for colon cancer. Constitutive activation of Signal Transducers and Activators of Transcription 3 (STAT3) signaling is frequently detected in human cancer including colon cancer, and has emerged as an attractive molecular target for cancer prevention and therapy. We tested the efficacy of inhibiting persistent STAT3 signaling by a dietary agent, Ursolic acid from basil and a novel STAT3-selective inhibitor, LY5 in human colon cancer cell lines. Our data demonstrated that Ursolic acid is able to inhibit STAT3 phosphorylation, and induce apoptosis in HCT116 and SW480 human colon cancer cell lines. These results were confirmed by LY5, a novel STAT3-selective small molecular inhibitor, which also inhibits STAT3 phosphorylation and induces apoptosis in HCT116 and SW480 colon cancer cell lines. In addition, LY5 inhibits IL-6 mediated induction of STAT3 phosphorylation in DLD-1 human colon cancer cells. We also found Ursolic acid decrease cell viability and inhibit the tumorsphere formation of ALDH+/CD133+ colon cancer stem cells. Furthermore, administration daily of Ursolic acid suppressed the HCT116 tumor growth in mice in vivo. In summary, our results suggest that STAT3 is target for colon cancer prevention and inhibition of persistently activated STAT3 using dietary agents or inhibitors may offer an effective prevention approach for colorectal carcinoma. Citation Format: Li Lin, Wenying Yu, Hui Xiao, Wenlong Wang, Chongqiang Zhao, Jiagao Lu, Chenglong Li, Jiayuh Lin. STAT3 as a cancer prevention target in colorectal cancer cells. [abstract]. In: Proceedings of the Eleventh Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2012 Oct 16-19; Anaheim, CA. Philadelphia (PA): AACR; Cancer Prev Res 2012;5(11 Suppl):Abstract nr B57.

Previous studies have shown that the novel microtubule poison, JG-03-14, which binds to the colchicine binding site of tubulin, has the capacity to kill breast tumor cells primarily through the promotion of autophagy. The current work was designed to determine whether autophagy was, in fact, the primary mode of action as well as susceptibility to JG-03-14 in two additional tumor cell models, the B16/F10 murine melanoma cell line and the HCT-116 human colon cancer cell line. Drug cytotoxicity was monitored based on viable cell number and clonogenic survival. Apoptosis was assessed by DAPI staining, the TUNEL assay and/or FACS analysis. Autophagy was monitored based on staining with acridine orange, redistribution and punctuation of RFP-LC3 and electron microscopy as well as p62 degradation. Senescence was evaluated based on β-galactosidase staining and alterations in cell morphology. Drug effects were also evaluated in a murine model of B16/F10 cells that localizes to the lungs while peripheral neuropathy was assessed by three complementary behavioral assays. Both HCT-116 colon cancer cells and B16/F10 melanoma cells were sensitive to JG-03-14 in that the drug demonstrated tumor cell killing. However, there was minimal induction of apoptosis. In contrast, there was clear evidence for autophagy and autophagic flux while the residual surviving cells appeared to be in a state of irreversible senescence. Inhibition of drug-induced autophagy in either the melanoma cells or the colon carcinoma cells was only slightly protective as the cells instead died by apoptosis. JG-03-14 reduced the size of tumor nodules in mice lungs; furthermore, the drug did not promote peripheral neuropathy. Taken together with evidence for its actions as a vascular disrupting agent, these observations support the potential utility of JG-03-14 to effectively treat malignancies that might be resistant to conventional chemotherapy through evasion of apoptosis.

To investigate the effects on telomerase activity of transfection of human T-STAR gene full-length sense cDNA or partial antisense cDNA into human colon cancer cell line HCT-116. mRNA and protein expression levels of T-STAR gene were determined by RT-PCR and western blot, and telomerase activity was measured by PCR-ELISA, after transfection of T-STAR sense or antisense gene into HCT-116 cells with lipofectamine. T-STAR gene expression was enhanced or knocked down both at mRNA and protein levels, and telomerase activity was significantly increased or decreased. The T-STAR gene may participate in regulation of telomerase activity in human colon cancer HCT-116 cells in a parallel fashion.

Objective To establish a human colon cancer cell line HCT-116/5-FU resistant to 5-fluorouracil(5-FU)and explore the relationship between runt-related transcription factor 3(RUNX3)and drug resistance of colorectal cancer.Methods The human colon cancer cell line HCT-116/5-FU with resistance to 5-FU was established by low concentration gradient increment combined with high-dose intermittent shock.CCK-8 method was used to determine the half maximal inhibitory concentration(IC50)of 5-FU on the parent line HCT-116 and drug-resistant line HCT-116/5-FU.The cell growth curve was established for the calculation of population doubling time(TD).The mRNA levels and protein levels of RUNX3,P-glycoprotein(P-gp),multidrug resistance-associated protein 1(MRP1),and lung resistance-related protein(LRP)in HCT-116 and HCT-116/5-FU cells were determined by qRT-PCR and Western blotting,respectively.The RUNX3 expression in HCT-116 cells was knocked down by siRNA technique,and the cells were divided into RUNX3 knockdown groups(si-RUNX3-1 group and si-RUNX3-2 group)and negative control group(si-NC group).The knockdown efficiency was verified by qRT-PCR at the mRNA level and Western blotting at the protein level.The IC50 in si-RUNX3 groups and si-NC group was determined with CCK-8 method,and the expression of P-gp,MRP1,and LRP in the two groups was detected by Western blotting.Results A stable human colon cancer drug-resistant cell line HCT-116/5-FU was successfully constructed.HCT-116/5-FU showed the TD 1.38 times as long as that of HCT-116(P=0.002)and changed morphology.The mRNA level of RUNX3 in HCT-116/5-FU cells was significantly lower than that in HCT-116 cells(P=0.048),and those of P-gp(P=0.008),MRP1(P=0.001),and LRP(P=0.001)showed the opposite trend.The protein level of RUNX3 in HCT-116/5-FU cells was significantly lower than that in HCT-116(P<0.001),and those of P-gp,MRP1,and LRP presented the opposite trend(all P<0.001).The HCT-116 cell model with low expression of RUNX3 was successfully established.The mRNA level of RUNX3 had no significant difference between si-RUNX3-1 group and si-NC group(P=0.064),while the level in si-RUNX3-2 group was significantly lower than that in si-NC group(P=0.034).The protein levels of RUNX3 in si-RUNX3-1 group and si-RUNX3-2 group were lower than that in si-NC group(both P<0.001).The results demonstrated higher knocking efficiency in si-RUNX3-2 group,which was thus selected to complete the follow-up test.The IC50 of si-RUNX3 group was significantly higher than that of si-NC group(P<0.001),which indicated that the down-regulated expression of RUNX3 could reduce the sensitivity of HCT-116 cells to 5-FU.The relative protein levels of P-gp,MRP1,and LRP in si-RUNX3 group were significantly higher than those in si-NC group(all P<0.001).Conclusion The down-regulation of RUNX3 expression can reduce the sensitivity of HCT-116 cells to 5-FU,which is considered to be related to the up-regulated expression of P-gp,MRP1,and LRP.

Stilbene 5c, a microtubule poison with vascular disrupting properties that induces multiple modes of growth arrest and cell death

Raddeanin A, one of the triterpenoid saponins extracted from Anemone raddeana rhizome of the Ranunculaceae family, has demonstrated the ability to inhibit the growth of human hepatic and gastric cancer cells. However, the effects of Raddeanin A on human colon cancer cells have not been investigated extensively. The present study aimed to examine the antiproliferative and apoptosis-inducing effects of Raddeanin A on the HCT-116 human colon cancer cell line in vitro, and evaluate the pharmacokinetic and biodistribution properties of Raddeanin A in mice following a single oral administration. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to assess the in vitro cytotoxicity of Raddeanin A against HCT-116 cells. 4',6-Diamidino-2-phenylindole, dihydrochloride staining and flow cytometry were performed to further examine the apoptosis-inducing capability of Raddeanin A. The concentrations of Raddeanin A in the plasma and tissues were analyzed using liquid chromatography-tandem mass spectrometry. Raddeanin A showed a dose-dependent antiproliferative effect towards the HCT-116 cells, with a half maximal inhibitory concentration of ~1.4 µM. Treatment with Raddeanin A resulted in a significant induction of apoptosis, observed as apparent morphological changes of the nuclei, with a total apoptotic ratio of 41.8% at a concentration of 3 µM. Low concentrations of Raddeanin A were detected in the heart, liver, spleen, lung, kidney and plasma of the mice following oral administration, however, the majority of the Raddeanin A was distributed in the intestinal tract, particularly in the colon and caecum. These present study confirmed the growth-inhibitory and apoptosis-inducing effects of Raddeanin A on HCT-116 cells and performed preliminary examinations of its pharmacokinetic properties, which provide a foundation for further investigating the inhibitory mechanism on the colon cancer cells in vivo.

Zamzam water (ZW) is a naturally hard alkaline type of water with unique physical and chemical properties that are different from any other water. The aim of the current work is to evaluate the potential anticancer activity of ZW against colon cancer. Human colon cancer HCT-116 and human skin fibroblast HSF cell lines were treated with two treatment conditions of ZW, Z1 with adjusted pH to 7.4 and Z2 without pH adjustment (pH 8). Cell viability was assessed using MTT and trypan blue dye exclusion assays. Cell cycle alterations and the type of cell death were investigated using flow cytometry technique. Cellular and mitochondrial reactive oxygen species (ROS) levels were quantified by H2DCFDA and MitoSOX assays, respectively. The results of the current study showed that both ZW treatments reduced cell viability of cancer cells. MTT assay showed a significant reduction in cell viability to 87% and 66%, respectively, after treatment with Z1 and Z2 (p≤0.0001). Cell death has occurred via apoptotic pathway under the two treatment conditions. The percentage of early apoptosis was 3%, 3.5% and 2.8% in control, Z1 and Z2 respectively. In the late apoptotic stage, there was a significant increase (4.2%) only for Z2 treatment in comparison to the control (p≤0.001). The cells were arrested in the G2/M phase of the cell cycle, and decreased in G1 phase after 24 hours of treatment with ZW. Only Z2 treatment, showed an increase in the production of both cytoplasmic and mitochondrial ROS. In conclusion, our results indicate, for the first time, that ZW induces apoptosis of human colon cancer HCT-116 cells, suggesting the potential anticancer effect of ZW against colon cancer.

Ganoderma lucidum polysaccharides (GLP) extracted from Ganoderma lucidum have been shown to induce cell death in some kinds of cancer cells. This study investigated the cytotoxic and apoptotic effect of GLP on HCT-116 human colon cancer cells and the molecular mechanisms involved. Cell proliferation, cell migration, lactate dehydrogenase (LDH) levels and intracellular free calcium levels ([Ca(2+)]i) were determined by MTT, wound-healing, LDH release and fluorescence assays, respectively. Cell apoptosis was observed by scanning and transmission electron microscopy. For the mechanism studies, caspase-8 activation, and Fas and caspase-3 expression were evaluated. Treatment of HCT-116 cells with various concentrations of GLP (0.625-5 mg/mL) resulted in a significant decrease in cell viability (P< 0.01). This study showed that the antitumor activity of GLP was related to cell migration inhibition, cell morphology changes, intracellular Ca(2+) elevation and LDH release. Also, increase in the levels of caspase-8 activity was involved in GLP-induced apoptosis. Western blotting indicated that Fas and caspase-3 protein expression was up-regulated after exposure to GLP. This investigation demonstrated for the first time that GLP shows prominent anticancer activities against the HCT-116 human colon cancer cell line through triggering intracellular calcium release and the death receptor pathway.

The synthesis of noble metal nanoparticles is currently experiencing substantial development and considerable attention. Plant extracts are commonly used for the biological synthesis of nanoparticles because they contain biologically active constituents. In our present study, silver nanoparticles (AgNPs) were synthesized using an aqueous Illicium verum (Star anise) extract to evaluate their antimicrobial, antioxidant, and cytotoxicity activities. For maximum yields of AgNPs, the extract (2.5 ml), silver ions (500 µM), and pH (8) were shown to be the ideal nanoparticle production parameters. The visual colour shifted from pale brown to dark brown when the ultraviolet-visible spectrophotometer was used to validate the synthesis of AgNPs. A transmission electron microscope was utilized to evaluate nanoparticles’ physical nature. The presence of silver metal with face-centred cubic symmetry was confirmed by X-ray diffraction analysis. Fourier-transform infrared spectroscopy was used to identify the functional groups in charge of reducing silver ions (Ag+) and the stability of AgNPs produced using the I. verum aqueous extract. The agar well diffusion method investigated the antibacterial activity of I. verum silver nanoparticles (Iv-AgNPs) against pathogenic bacteria and fungi. At higher doses (100 µg·mL−1), the highest zone of inhibition was observed, and spherical AgNPs demonstrated the antibacterial activity. The I. verum extract and Iv-AgNPs enhanced (70%) their free radical scavenging activity at 500 µg·mL−1 according to the 2,2-diphenyl-1-picrylhydrazyl assay. Moreover, the cytotoxicity of Iv-AgNPs against the HCT-116 human colon cancer cell line indicated cell inhibition in a dose-dependent manner. Ultimately, the findings of this study indicate that techniques used to produce AgNPs are environmental friendly, cost-effective, harmless, uncomplicated, and can effectively tackle a broad spectrum of medical and nutritional concerns.

Heilaohu, the roots of Kadsura coccinea, has a long history of use in Tujia ethnomedicine for the treatment of rheumatoid arthritis and gastroenteric disorders, and a lot of work has been done in order to know the material basis of its pharmacological activities. The chemical investigation led to the isolation and characterization of three new (1–3) and twenty known (4–23) lignans. Three new heilaohulignans A-C (1–3) and seventeen known (4–20) lignans possessed dibenzocyclooctadiene skeletons. Similarly, one was a diarylbutane (21) and two were spirobenzofuranoid dibenzocyclooctadiene (22–23) lignans. Among the known compounds, 4–5, 7, 13–15 and 17–22 were isolated from this species for the first time. The structures were established, using IR, UV, MS and NMR data. The absolute configurations of the new compounds were determined by circular dichroism (CD) spectra. The isolated lignans were further evaluated for their cytotoxicity and antioxidant activities. Compound 3 demonstrated strong cytotoxic activity with an IC50 value of 9.92 µM, compounds 9 and 13 revealed weak cytotoxicity with IC50 values of 21.72 µM and 18.72 µM, respectively in the HepG-2 human liver cancer cell line. Compound 3 also showed weak cytotoxicity against the BGC-823 human gastric cancer cell line and the HCT-116 human colon cancer cell line with IC50 values of 16.75 µM and 16.59 µM, respectively. A chemiluminescence assay for antioxidant status of isolated compounds implied compounds 11 and 20, which showed weak activity with IC50 values of 25.56 µM and 21.20 µM, respectively.