Cytotoxicity and reversal of multidrug resistance by tryptanthrin-derived indoloquinazolines.

AIM: To evaluate the effects and elucidate the mechanisms of a series of indoloquinazolines as novel anticancer agents. METHODS: Condensation of the substituted isatoic anhydride with the substituted isatin was performed to prepare compounds 1-4, followed by adding malononitrile to prepare compounds 5-7. Cytotoxicity was measured by MTT assays. Apoptosis induction was evaluated using DNA fragmentation, cell cycle assay, caspase 3/7 activity and Western blot. RESULTS: Compounds 3, 4, and 5 display cytotoxicity against MCF-7, HeLa, SKOV3, and A498 cancer cells. DNA ladders appear in cells treated with compounds 3, 4, and 5. Within those, compound 4 exhibits the greatest activity in regards to sub-G(1) accumulations in the cell cycle and the activation of caspase-3/7. Furthermore, Fas and Fas ligand levels are elevated by compound 4, implying that the apoptosis is in part mediated through the signals. On the other hand, compounds 1 and 7 display chemosensitizing activity since cytotoxicity of doxorubicine and etoposide is enhanced in combination with compound 1 and 7, respectively, in MCF-7/adr (doxorubicin-resistant) and MCF-7/vp (etoposide-resistant). CONCLUSION: The cytotoxicity of indoloquinazolines is structure-dependent rather than cell type-dependent due to the similar degree of cytotoxicity induced by the individual compounds in all four cell lines. Further modification of the tryptanthrin skeleton is important to develop novel anticancer agents bearing either cytotoxicity against MCF-7 cells or drug resistance reversal in MCF-7/adr and MCF-7/vp.

Downregulation of GSTpi expression by tryptanthrin contributing to sensitization of doxorubicin-resistant MCF-7 cells through c-jun NH2-terminal kinase-mediated apoptosis.

Overexpression of GSTpi and underexpression of Topo II expression are associated with multidrug resistance (MDR) phenotype through nontransporter pathway. Tryptanthrin, a quinazoline derivative, was reported to sensitize resistant cells to doxorubicin by downregulation of MDR1 expression. This study aims to extendedly investigate the effect of tryptanthrin on the role of nontransporter-based genes in determining the MDR response in doxorubicin-resistant MCF-7 cells (MCF-7/adr). Results show that tryptanthrin downregulates GSTpi expression and reduces glutathione S-transferase (GST) activity, but has no effect on Topo II expression. Less production of GSTpi decomposes the protein-protein interactions of GSTpi and c-jun NH2-terminal kinase (JNK). The resulting free-form JNK undergoes phosphorylation upon elevated intracellular doxorubicin accumulation and subsequently activates JNK-mediated apoptosis. In conclusion, in addition to transporter pathway, tryptanthrin reverses MDR partly by modulating GSTpi-related pathway, a nontransporter pathway, in MCF-7/adr cells. It indicates that tryptanthrin may act as a potential chemoadjuvant agent through multiple targets.

Apoptosis and necrosis are involved in the toxicity of Sauropus androgynus in an in vitro study.

BACKGROUND/PURPOSE: The raw juice of the young sticks and leaves of Sauropus androgynus (SA) has been widely used as a natural food for body weight reduction and vision protection in Taiwan and Southeast Asia. But as has been reported, SA-associated obliterative bronchiolitis can develop after taking SA for more than 3 months. Lung transplantation was carried out in severe cases. METHODS: To study the toxic effect, we separated the SA extract into three parts, namely CHCl3, EtOAc and n-BuOH fractions, using polarity dissection. NIH3T3 fibroblasts were treated with the SA fractions 300 microg/mL and subjected to a series of cytotoxic assays. RESULTS: The EtOAc fraction exhibited the strongest effect of cell growth inhibition, followed by the CHCl3 and n-BuOH fractions. Features of condensed chromatin and apoptosis were observed in cells exposed to n-BuOH and EtOAc fractions using fluorescence microscopy. Formation of DNA ladders was also observed in the above cells. Instead, the CHCl3 fraction induced DNA smearing. In bivariate dot plots of annexin V and propidium iodide double staining, necrosis and apoptosis appeared in cells treated with CHCl3 and n-BuOH fractions, respectively, and a mixed type of necrosis and apoptosis appeared in EtOAc fraction-treated cells. CONCLUSION: Our results indicate that necrosis and apoptosis are involved in the toxic effect of SA in NIH3T3 fibroblasts. More evidence is needed to clarify if necrosis and apoptosis are also related to the pathogenesis of SA-associated obliterative bronchiolitis.

Gain-of-function p53 mutant with 21-bp deletion confers susceptibility to multidrug resistance in MCF-7 cells.

The majority of p53 mutations, which are responsible for gain of oncogenic function, are missense mutations in hotspot codons. However, in our previous study, we demonstrated that a deletion spanning codons 127-133 in the p53 gene (designated as del p53) was detected in doxorubicin-resistant MCF-7 cell lines following various induction processes. In the present study, we aimed to investigate the role of del p53 and its association with the proliferation, metastasis and drug resistance of MCF-7 cells. The MCF-7/del p53 cell line is a representative of the del p53 stably expressed clones which were constructed by transfection of the del p53-containing construct into MCF-7/wt cells. Markers of multidrug resistance (MDR), epithelial-mesenchymal transition (EMT) and stem cell-like properties were examined in the MCF-7/del p53 cells. The results revealed that the MCF-7/del p53 cells expressed full-length p53 and del p53 mRNA and protein, as well as P-glycoprotein (P-gp). The MCF-7/del p53 cells acquired resistance to doxorubicin with increased P-gp efflux function. Using a transient expression assay, the mdr1 promoter was found to be significantly activated by external or integrated del p53 (P<0.001). The inhibition of nuclear factor (NF)-κB by cyclosporine sensitized the MCF-7/del p53 cells to doxorubicin toxicity. In addition, the morphological characteristics of the MCF-7/del p53 and MCF-7/adr were similar. EMT was observed in the MCF-7/del p53 cells as demonstrated by the presence of the mesenchymal markers, Slug and vimentin, and the decrease in the epithelial marker, cadherin 1, type 1, E-cadherin (CDH1), as well as an enhanced migration ability (P<0.001). Furthermore, the number of cells expressing the cancer stem cell-like marker, CD44, increased, accompanied by mammosphere formation. Taken together, these findings indicate that the expression of del p53 in MCF-7/del p53 cells enables the cells to partially acquire doxorubicin resistance characteristics of the MCF-7/adr cells. Thus, del p53 may be an important factor in non-invasive MCF-7 cells, activating NF-κB signaling and the mdr1 promoter and partially attributing to EMT; the cells thus acquire stem cell­like properties, which facilitates drug resistance. Therefore, the 21-bp deletion of p53 may prove to be a therapeutic strategy with which to prevent cancer cells from acquiring resistance to drugs.

A critical dose of doxorubicin is required to alter the gene expression profiles in MCF-7 cells acquiring multidrug resistance.

Cellular mechanisms of multidrug resistance (MDR) are related to ABC transporters, apoptosis, antioxidation, drug metabolism, DNA repair and cell proliferation. It remains unclear whether the process of resistance development is programmable. We aimed to study gene expression profiling circumstances in MCF-7 during MDR development. Eleven MCF-7 sublines with incremental doxorubicin resistance were established as a valued tool to study resistance progression. MDR marker P-gp was overexpressed only in cells termed MCF-7/ADR-1024 under the selection dose approaching 1024 nM. MCF-7/ADR-1024 and authentic MCF-7/ADR shared common features in cell morphology and DNA ploidy status. MCF-7/ADR-1024 and authentic MCF-7/ADR down regulated repair genes BRCA1/2 and wild type p53, apoptosis-related gene Bcl-2 and epithelial-mesenchymal transition (EMT) epithelial marker gene E-cadherin. While detoxifying enzymes glutathione-S transferase-π and protein kinase C-α were up-regulated. The genes involving in EMT mesenchymal formation were also overexpressed, including N-cadherin, vimentin and the E-cadherin transcription reppressors Slug, Twist and ZEB1/2. PI3K/AKT inhibitor wortmannin suppressed expression of Slug, Twist and mdr1. Mutant p53 with a deletion at codons 127-133 markedly appeared in MCF-7/ADR-1024 and authentic MCF-7/ADR as well. In addition, MCF-7/ADR-1024 cells exerted CSC-like cell surface marker CD44 high/CD24 low and form mammospheres. Overall, results suggest that resistance marker P-gp arises owing to turn on/off or mutation of the genes involved in DNA repair, apoptosis, detoxifying enzymes, EMT and ABC transporters at a turning point (1.024 µM doxorubicin challenge). Behind this point, no obvious alterations were found in most tested genes. Selection for CSC-like cells under this dose may importantly attribute to propagation of the population presenting invasive properties and drug resistance. We thereby suggest two models in the induction of drug resistance. Model 1: Selection for CSC-like cells. Model 2: Mutations for gain-of resistance. Either model 1 or model 2 requires doxorubicin dose approaching 1 µM to alter gene regulation.

Tryptanthrin inhibits MDR1 and reverses doxorubicin resistance in breast cancer cells.

Development of agents to overcome multidrug resistance (MDR) is important in cancer chemotherapy. Up to date, few chemicals have been reported to down-regulate MDR1 gene expression. We evaluated the effect of tryptanthrin on P-glycoprotein (P-gp)-mediated MDR in a breast cancer cell line MCF-7. Tryptanthrin could depress overexpression of MDR1 gene. We observed reduction of P-gp protein in parallel with decreases in mRNA in MCF-7/adr cells treated with tryptanthrin. Tryptanthrin suppressed the activity of MDR1 gene promoter. Tryptanthrin also enhanced interaction of the nuclear proteins with the negatively regulatory CAAT region of MDR1 gene promoter in MCF-7/adr. It might result in suppression of MDR1 gene. In addition, tryptanthrin decreased the amount of mutant p53 protein with decreasing mutant p53 protein stability. It might contribute to negative regulation of MDR1 gene. In conclusion, tryptanthrin exhibited MDR reversing effect by down-regulation of MDR1 gene and might be a new adjuvant agent for chemotherapy.

Olanzapine induces SREBP-1-related adipogenesis in 3T3-L1 cells.

Olanzapine is a second-generation atypical antipsychotic drug (AAPD). Major side effects of olanzapine are weight gain and development of diabetes mellitus, which are risk factors of cardiovascular diseases. The possible causes of metabolic adverse effects are known as poor satiety and increased food intake due to blockade of receptors such as 5-HT(2C) in CNS. In this study, we examine the effect of olanzapine on peripheral adipogenesis using cultured 3T3-L1 cell model. Olanzapine increased triacylglyceride (TG) accumulation during 3T3-L1 preadipocyte differentiation to mature adipocyte phenotype. TG accumulation was accompanied by overexpression of fatty acid synthase and adiponectin that are the downstream genes of sterol regulatory element binding protein-1 (SREBP-1), one of the key transcription factors in lipid homeostasis. We further consisted that mostly SREBP-1 and at a lesser extent peroxisome proliferator-activated receptor gamma (PPAR-gamma), but not CCAAT/enhancer binding protein-alpha (C/EBP-alpha), were overexpressed and activated in 3T3-L1 adipocytes exposed to olanzapine. Furthermore, we showed that olanzapine enhanced the activity of SRE-1-containing LDLR promoter in transfected 3T3-L1 adipocytes and HepG2 cells. Taken together, olanzapine may cause body weight gain not only through influencing CNS receptors, but also affecting the peripheral adipogenesis regulated by SREBP-1.

3-O-beta-D-glucosyl-(1-->6)-beta-D-glucosyl-kaempferol isolated from Sauropus androgenus reduces body weight gain in Wistar rats.

The young sticks and leaves of Sauropus androgynus (SA) that had been used as a health food for body weight reduction, led to an outbreak of obliterative bronchiolitis in Taiwan. This study tested the toxicity and anti-obesity features of the SA-isolated compound, 3-O-beta-D-glucosyl-(1-->6)-beta-D-glucosyl-kaempferol (GGK), on male Wistar rats receiving 6 or 60 mg/kg of GGK orally as well as partial purified EtOAc and n-BuOH fractions of SA extract daily for 28 d. Sixty milligrams per kilogram GGK treatment significantly reduced food intake in rats by 15% (p<0.05). The reduced food intake corresponded to decreases in body weight in the high or low dose GGK groups, as compared to the control groups. The serum levels of free triglyceride significantly decreased in GGK-treated rats. GGK treatment led to succesive reductions in daily food intake and body weight without obvious histopathological changes in Wistar rats. Thus, GGK may be potentially to be developed as a safe and novel compound for anti-obesity treatment.