Chemosensitizing activities of cyclotides from Clitoria ternatea in paclitaxel-resistant lung cancer cells

Cyclotides comprise a family of circular mini-peptides that have been isolated from various plants and have a wide range of bioactivities. Previous studies have demonstrated that cyclotides have antitumor effects and cause cell death by membrane permeabilization. The present study aimed to evaluate the cytotoxicity and chemosensitizing activities of cyclotides from Clitoria ternatea in paclitaxel-resistant lung cancer cells. In this study, a total of seven cyclotides were selected for colorimetric cell viability assay (MTT assay) to evaluate their anticancer and chemosensitizing activities in the lung cancer cell line A549 and its sub-line A549/paclitaxel. Results suggested that certain cyclotides had significant anticancer and chemosensitizing abilities; such cyclotides were capable of causing multi-fold decreases in the half maximal inhibitory concentration (IC50) value of cliotides in the presence of paclitaxel. More importantly, their bioactivities were found to be correlated with their net charge status. In conclusion, cyclotides from C. ternatea have potential in chemosensitization application.


Introduction
An abundance of cyclotides have been isolated and confirmed from Clitoria ternatea (Fabaceae family) (1,2). The concentration of cyclotides from this plant is relatively higher than that of the majority of cyclotide-containing plants from the Rubiaceae and Violacecae families. C. ternatea is there-fore an ideal resource for investigating the bioactivities of cyclotides. In previous studies, in vitro cell-based assays have demonstrated that certain cyclotides have significant cytotoxic activities by disrupting the cell membrane integrity (3,4). By contrast, in an in vivo study, even the most potent cyclotide, cycloviolacin O2, did not demonstrate a significant anticancer effect in a xenograft model (5). Gerlach et al revealed that cyclotides may have potential chemosensitizing abilities when combined with other anticancer reagents (6). Cycloviolacin O2 and psyle cyclotides from Psychotria leptothyrsa were used to treat the doxorubicin-resistant human breast cancer cell line MCF-7/ADR. It was found that cyclotides were capable of enhancing the doxorubicin effect that inhibits MCF-7/ADR proliferation; coexposure caused the half maximal inhibitory concentration (IC 50 ) value to decrease 4-to 7-fold compared with single doxorubicin. The mechanism was considered to be associated with a leakage of cell membrane caused by cyclotides, thus enabling the chemical reagent to pass through the cell membrane. Therefore, we hypothesize that this type of chemosensitizating ability may be wide spectrum with less selectivity. In the present study, the human lung cancer cell line A549 and its sub-line A549/paclitaxel were selected to provide support for this hypothesis. The role of the net charge status of cyclotides on cytotoxicity and chemosentization was also investigated.

Materials and methods
Isolation and purification of cyclotides. Cyclotides from C. ternatea, termed specifically as cliotides, were isolated and purified from C. ternatea as described previously (2). Briefly, flowers and seeds were ground, and then extracted by 20% ethanol (V/V, with water). Crude extract was centrifuged for 30 min at 10,000 x g and the supernatant was passed through a 0.45 µm filter to clear the debris. Different types of cliotide peptides with different acetonitrile concentration gradients were fractionated and purified by RP-HPLC. Based on the amino acid sequence and net charges, a total of seven cliotide peptide molecules were selected for use in the present study (Table I).
Cell culture. Human non-small lung cancer (A549) cells were obtained from the American Type Culture Collection Cytotoxicity assay. Various types of cliotides were assessed for their cytotoxicity activity in A549 cells and their chemosensitizating capability in A549/taxol cells, as previously described by Gerlach et al (6). Briefly, cells were seeded in 96-well flatbottomed microtiter plates (1x10 4 ). After culture for 4 h, cells were treated with cliotide T2 (CT2), CT4, CT7, CT10, CT12, CT19, CT20 and paclitaxel at 0.4, 1, 2, 4, 10 and 20 µM, in 100 µl of media for 72 h. The medium was removed from each well and 100 µl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT; 0.5 mg/ml in PBS) was added in the absence of light; formazan crystals were produced over a 4 h incubation period. To dissolve crystals, 150 µl of 0.04 N HCl in isopropanol was added to each well and the optical density at 540 nm was measured on a Tecan Spectrophotometer (Tecan SPECTRAFluor, Tecan, Männedorf, Switzerland). For coexposure experiments, the same protocol was followed but concentrations were modified to 50:50 ratios of each cliotide and paclitaxel at the following concentrations: 0.2, 0.5, 1, 2, 5 and 10 µM. Each test was performed with 3 replicates and repeated in triplicate. Replicates were averaged and the blank subtracted from control and test concentrations. Optical density of the treated wells was compared with that of the controls (100% survival); percentage cell survival was calculated and plotted by GraphPad Prism (GraphPad Software, Inc.; San Diego, CA, USA).
Statistical analysis. All data were analyzed with GraphPad Prism 4.0 software. The IC 50 values (µM) of each treatment were calculated and a combination of paired t-tests and analysis of variance (ANOVA) were performed. P<0.05 was considered to indicate a statistically significant difference. Table I were used to test cytotoxicity in A549 cells. All of these demonstrated significant cytotoxicity, with the exception of CT19 and CT20 whose predicted IC 50 values were ~10 µM (Fig. 1). Previous studies have suggested that net charge is an important factor in cytotoxic activity, and bracelet cyclotides were generally more cytotoxic than Möbius cyclotides (7). In the present study, CT4, which had a net charge of +2, had the lowest IC 50 value (0.21 µM) compared with the other cyclotides. However, a positive charge of >2 did not increase the cytotoxicity; CT19 had a charge of +3 and its IC 50 value was ~10 µM, while CT20 had a charge of +4, with an IC 50 value above 10 µM.

Cytotoxicity of cliotides. All cliotides in
Chemosensitization of cliotides with paclitaxel on A549/paclitaxel. Based on the results above, cliotides which had significant cytotoxicity in A549 cells (IC 50 <10 µM) were selected for coexposure experiments in the A549 paclitaxel-resistant cell line. These cliotides were CT2, CT4, CT7, CT10 and CT12. Fig. 2A-E and Table II summarize the chemosensitizing effect of five types of cliotide, all of which demonstrated positive results reflected by the multi-fold decreases in the IC 50 value of cliotides in the presence of paclitaxel. A four-fold decrease in the CT2-treated group, a three-fold decrease in the CT4-, CT7-and CT10-treated groups and a one-fold decrease in the CT12-treated group were observed.

Discussion
At present, >200 cyclotides have been isolated and identified from various plants of the Violaceae, Rubiaceae, Cucurbitaceae and Fabaceae families (8-11). Cyclotides comprise a family of circular mini-proteins and have a wide range of bioactivities. They have a characteristic head-to-tail cyclized backbone typically composed of 28-37 amino acids, and a knotted disulfide topology involving six conserved cysteine residues (12-14). The cyclic backbone and conserved cysteine residues form a unique

A B
C D E structure termed the cyclic cystine knot (CCK), in which two of the disulfide bonds and their connecting backbone segments form an embedded ring that is penetrated by the third disulfide bond (14). Aside from being a characteristic structural feature of the cyclotide family, the CCK motif enables these miniproteins to be exceptionally resistant to chemical, enzymatic and thermal degradation (15). Therefore, cyclotides have become an ideal scaffold for drug delivery (15,16). Cyclotides exhibit a range of biological activities, including uterotonic (17), hemolytic (18), antineurotensin (19), anti-HIV (20), cytotoxic (3) and antibacterial (18) activities; however, their natural function is considered to be as plant defence molecules, based on their insecticidal (21) and molluscidal (22) properties. Among all the bioactivities of cyclotides, cytotoxicity was was one of the first to be identified and has been investigated widely. However, the sharp dose-response profile and poor in vivo anticancer results affected the usage of cyclotides in clinical treatment (5). In the present study, consistent with previous research, cliotides were less cytotoxic against A549/paclitaxel compared to A549 (Table II). Additionally concordant, cliotides with a charge of +2 were the most cytotoxic; CT4 had the lowest IC 50 value. The cytotoxic ranking was IC 50 CT4 (+2)<IC 50 CT7 (+1) and CT12 (+1)<IC 50 CT10 (0)<IC 50 CT2 (-1); cytotoxicity increased with an increase in net positive charge. However, CT19 and CT20, which had charges of +3 and 4, respectively, did not show significant cytotoxicity. With regard to why higher cliotide positive charges may decrease cytotoxicity, we hypothesized that although multiple positive charges made it easier for the cliotides to adhere to the cell membrane, they also changed the peptide space structure and reduced the hydrophobicity, thus reducing the ability to disrupt the cell membrane.
Drug resistance is one of the greatest obstacles limiting chemotherapy in clinical treatment. The chemosensitizing ability of the cliotides in the A549/paclitaxel cells in the present study is notable; all five tested cyclotides demonstrated a two-to four-fold decrease in IC 50 value in the presence of paclitaxel.
In summary, this study demonstrated that cyclotides from C. ternatea have the potential to be used in chemosensitization for treating cancer. We are optimistic that cyclotides are suitable drug candidates after modification and their charge status plays a significant role in their bioactivities.