Abstract
Curcin, a type 1 ribosome inactivating protein (RIP) is investigated here for its cellular competence on six mammalian cell lines. Cells exposed to curcin (100 μg/ml) for 72 h exhibited significant cellular metabolic arrest, with the cancer cell lines being more sensitive. The viability assessment of the cancer cells in a 3D cell culture based assay revealed highly restricted sprouting and proliferation with near to complete dead cell population. Prominent mitochondrial dysfunction, elevated reactive oxygen species levels, nuclear degeneration, structural/mechanical destabilization and suppression of defense mechanisms were imminent with the RIP treated cells. Expression levels of nuclear factor κB (NF-κB), cytoskeletal focal adhesion kinases (FAK) and vinculin were significantly diminished. Vital cellular organelles as nucleus, mitochondria and actin were severely incapacitated on RIP exposure resulting in multimodal apoptosis and necrosis. The ability of curcin to impart comprehensive shutdown of the cells, especially cancer cells, complemented with its hemocompatibility, opens up possibilities of utilizing this ribotoxin as a prospective therapeutic candidate against cancers of diverse origins.
Similar content being viewed by others
Introduction
Phytomolecules have long served a pivotal role as potent active components towards strengthening and widening the pharmaceutical options against various ailments. Ribosome inactivating proteins (RIPs) are one such highly cogent group of proteins, mainly of plant, bacterial and fungal origin, which have conventionally hosted the ability to irreversibly depurinate the ribosomal RNA and terminate protein synthesis1,2,3. Traditionally, RIPs are recognized to deadenylate the large ribosomal RNA with high site specificity and inactivate the ribosomes owing to their RNA N-glycosidase activity4,5,6. The potential of some of the RIPs to depurinate the rRNA at multiple sites7 along with several other polynucleotide substrates8 has been addressed. Broad and diverse pharmacological attributes have been associated with RIPs, as protein synthesis-inhibitory, immunosuppressive, anti-tumor, antiviral, anti-HIV and abortificient traits to mention a few9.
The fascinating multimodal activities of RIPs have since long time generated interest towards developing antitumor drugs that selectively target tumor cells10. A stern resolve in this regard, particularly in the case of type 1 RIPs, are the immunotoxins11,12,13,14,15, RIPs conjugated to specific antibodies, which may present promising options for treating various diseases and interestingly against HIV-infected cells16. The action mechanism of RIPs have been extremely imperceptible and numerous accounts have been put forth to decipher the same. It was earlier believed that the mortality of cells treated with RIPs or RIP-immunotoxins was a result of protein synthesis inhibition alone with subsequent cellular necrosis, however, a stark similarity between RIP treated cells and cells undergoing apoptosis or programmed cell death (PCD) has been observed17,18,19,20,21.
Jatropha curcas is a well-known and mass cultivated multipurpose plant with immense medicinal value22,23,24. Felke25 isolated the toxic principle curcin, from the seeds of this plant which is a type 1 RIP possessing strong inhibitory activity on protein synthesis1 along with N-glycosidase and antitumor activities4,26,27,28. Recent works have demonstrated the cancer directed therapeutic potential of this RIP in conjunction with peptides and nanoparticles29,30, additionally emphasizing the compatibility of curcin to collaborate with various delivery strategies without compromising on its activity.
In this study, we explore the superior toxicity of curcin against normal and cancer cell lines. Morphological events related to organelle degeneration and membrane protein disruptions during apoptosis have also been described with an emphasis on the inherent ability of curcin to affect Nuclear factor κB (NF-κB), Focal adhesion kinases (FAK), Poly (ADP-ribose) polymerase (PARP) and reactive oxygen species (ROS). Further, the toxic effects of curcin on 3D in vitro cancer mass was assayed and recorded, disrupting and destroying the cancer colonies with intense proficiency. In addition, suppression of the anti-apoptotic survivin and the extra cellular matrix binding vinculin proteins, have been evidenced correlating the multiple modes of action of this potent phytomolecule to induce apoptotic stimuli and consequent cytocidal reactions.
Results
Cytocidal effects of curcin
The level of cellular metabolic activity greatly depends on proper functioning of organelles and any dysfunction leads to impaired cyto-metabolism. With respect to the cell lines under study, cytocidal rather than cytostatic effects of curcin were observed. Dose dependent toxicity was recorded, with the severity increasing with increase in curcin concentration (Fig. 1A). In the case of normal cell lines, L929, HCN-1A and HuVEC at the highest concentration of curcin (100 μg/ml), approximately 70%, 35% and 60% of cells, respectively, were viable. The fibroblast L929 has a higher mitotic index when compared to the other two normal cell lines, which may be a factor in the relatively increased viability. Endothelial cells, though with lower mitotic index, showed only moderate response to curcin treatment. This could be due to the efflux transport mechanism these cells exhibit as part of being in vascular system, exporting molecules across tissues and blood stream.
The effect of curcin on cancer cells was profound with glioma registering below 20% viability at 100 μg/ml and about 65% at 1 μg/ml of the RIP. The mortality rate of MCF-7 was nearly 70% at 100 μg/ml of curcin and around 45% at 10 μg/ml. Though susceptible, yet the most resistant among the cancer cell lines was the multi-drug resistant MDA-MB-453 presenting around 45% viability at the highest and nearly 55% at the mid concentration of curcin. As these cell lines possess more or less similar mitotic index, the variance in sensitivity to curcin may be linked to their drug resistance, which is unique to each cell population. We speculate that less sensitive MDA-MB-453, which is a triple negative multi-drug resistant variant, exhibited better drug efflux when compared to the other cancer cell lines. Glioma's increased sensitivity to curcin shows that it exhibits nil or minimal drug resistance.
Curcin treated cells predominantly exhibited apoptotic and necrotic characteristic events at hypodiploid regions, in many cases both signals recorded from same cells, invoking the complexity of toxicity rendered by the toxin (Fig. 1B). These observations were emulated in the flow cytometry (Supplementary Fig. S2) analysis, with curcin treated populations exhibiting prominent apoptosis and necrosis when compared to the controls.
To visually ascertain the metabolic arrest of cells treated with curcin, a new and simple approach based on the alamar blue cytotoxicity assay was attempted. The alamarBlue® Assay incorporates a fluorometric growth indicator based on detection of metabolic activity. Specifically, the system incorporates an oxidation-reduction (REDOX) indicator that both fluoresces and changes color in response to chemical reduction of growth medium resulting from cell growth. The underlying principle involves the reduction of non-fluorescent resazurin to a fluorescent dye, resarufin by metabolically active cells, which is not possible with cells whose metabolic activities have been terminated. Correspondingly, control cells showed highly intense fluorescent signals (Fig. 1Cg–l) which is directly proportional to cell's viability and its metabolic activity. In the case of curcin treated cells, nil or extremely weak intensity of fluorescence was recorded (Fig. 1Cs–x), translating directly to the diminished metabolic potential of the cells. The arrested metabolic state of the cells provides insight into the intrinsic stress that cells undergo due to exposure to toxins as curcin. To emphasize on the potential significance of this method, specifically dead cell population or cells undergoing apoptosis was concentrated upon in the case of curcin treated cells, which enables the enhanced discrimination potential of the metabolically active and dead cells.
In addition, corresponding morphological analysis revealed that curcin treated cells showed specific similarities to those undergoing a suicidal apoptotic fate. Visual indications included cellular content shrinkage, shriveling, membrane blebbing, plasma membrane integrity loss and clumping of cells, loss of organelle recognition and appearance of apoptotic bodies, among others.
In vitro 3D cancer colony regression and extirpation
The role of curcin, as a cancer repressor was analyzed with 3D colony assay. Cancer cells grow as 3D colonies, resembling tumor mass, on gel matrices. Accordingly, glioma developed into a 3D spheroid with the main colony being surrounded by smaller ones (Fig. 2a1), whereas MCF-7 and MDA-MB-453 grew as multi layered cellular mats (Fig. 2b1,c1). 96 h curcin treated groups showed decreased colony area (Fig. 2a4) and formation of elongated tubes projecting outwards of the cancer mass (Fig. 2d1,d2,e1,e2). Glioma spheroid remained as a dead mass of cells (Fig. 2a4) which kept on shrinking in due progression to curcin exposure and finally disintegrated. MCF-7 and MDA-MB-453 cancer masses were highly disrupted and reduced to tiny patches (Fig. 2b4,c4) associated with each other by thick tubular protrusions (in the case of MCF-7, Fig. 2e1,e2) which eventually disintegrated (Fig. 2b4). Also, majority of the treated cells had undergone an apoptotic or necrotic fate (Fig. 2a6,b6,c6). Calcein-AM, acetoxymethyl ester of calcein, is highly lipophilic and cell membrane permeable. Though Calcein-AM itself is not a fluorescent molecule, the calcein generated from Calcein-AM by esterase in a viable cell emits strong green fluorescence differentiating live and dead cells. On the other hand, PPI, a nuclei staining dye, cannot pass through a viable cell membrane. It reaches the nucleus by passing through disordered areas of dead cell membrane and intercalates with the DNA double helix of the cell to emit red fluorescence. The observations based on the fluorometric assay were in line with this principle, correlative of the cell membrane disruptions and related apoptotic/necrotic events on curcin exposure. The cancer colonies failed to propagate in culture even after regular media supplementation, evidencing the loss of sustenance and proliferation potential of the cells. These results reemphasize the profound detrimental effects administered by curcin to monolayer as well as multi-layered 3D carcinomas.
ROS generation and mitochondrial response
ROS triggering ability of curcin was complimentary to the cell viability data, reiterating the dose dependent activity of curcin. In normal and cancer cells alike, ROS was significantly more than that of control group. L929 cells showed an ROS generation of nearly 50% more than the control cells whereas HCN-1A and HuVEC cells produced a little more than 10 and 15% of the amount of ROS of control group, respectively. The cancer cells in particular exhibited enhanced ROS generation tendencies at all the concentrations of curcin tested, glioma and MDA-MB-453 being more responsive. Glioma cells presented an ROS capacity of nearly double that of the untreated cells. Meanwhile MCF-7 and MDA-MB-453 cells showed an increase in ROS of 40% and 75% respectively (Fig. 3A).
To analyze the secondary effects of ROS, immediate mediator of ROS-induced cell death mediated signaling, NF-κB activation was profiled. Increased absorbance intensity of NF-κB was observed in test groups (Fig. 3B). In the case of normal cell lines, L929, HCN-1A and HuVEC, the amount of activated NF-κB was slightly higher than controls whereas, glioma showed nearly twice the intensity of NF-κB compared to control. MCF-7 and MDA-MB-453 also presented noticeable increase in NF-κB levels.
Also, it could be clearly observed from Fig. 3C that the curcin treated cells showed diminished or any calcein fluorescence (Fig. 3Ca5,b5,c5,d5,e5,f5), depicting the leaching out of dye from mitochondrial pores, induced as a result of the increased ROS load in the mitochondria. In addition, loss of prominent fluorescence and patched signals in the cytosol portrayed the severe damage to mitochondrial membrane (Fig. 3Ca6,b6,c6,d6,e6,f6), induced as a result of toxin exposure. Additional prominent loss of mitochondrial integrity (Supplementary Fig. S3) has also been provided as concrete proof of the imminent toxicity of curcin on the cell's energy hub.
Nuclear deterioration and PARP inhibition
Significant nuclear deterioration was observed with cells exposed to curcin. The effects were prominent in 72 h curcin incubated cells with the formation of micronucleus, nuclear shrinkage, condensation of nuclear material (Fig. 4Ab4,d4,f4,h4,j4,l4) and total loss of morphological integrity (Fig. 4Aa4,c4,e4,g4,i4,k4). Time dependent progression of nuclear deformities with increase in curcin incubation was evident from the observations. The poly (ADP-ribose) polymerase (PARP) family of enzymes plays a critical role in the maintenance of DNA integrity as part of the base excision pathway of DNA repair. The results clearly indicated that curcin inhibited PARP by nearly 45% (Fig. 4B) at highest concentration and the inhibition rate was concentration dependent. PARP inhibition leads to cells being prone to DNA damage consequently switching on the DNA damage mediated apoptotic pathway, correlative of the observations with nuclear distortions on curcin exposure.
Cytoskeletal protein degeneration
Visual observations of curcin treated cells revealed a major structural and morphological transition from the normal (untreated) cells, with cytoplasmic shrinkage (Fig. 5m–r) and microtubule like outgrowths (Fig. 4k3,i2), highly arrested neuritic and dendritic structures (Fig. 6o) and eventual complete cellular collapse. An analysis of the cytoskeletal protein actin revealed that control groups devoid of curcin presented well-organized and elaborate actin network scaffolding throughout the cytoplasm (Fig. 5Ag–l) whereas cells treated with curcin showed prominent actin filament degeneration and distortion (Fig. 5As–x). The fine network of actin filaments were reduced to circumferential lining (Fig. 5Av) or to highly condensed matter (Fig. 5At, w, x). These results directly correlate to the morphological changes observed with curcin treated cell lines due to structural integrity loss. The expression levels of FAK, one of the main signaling molecules involved in variety of actin-mediated signaling pathways, in test group of cells decreased when compared to the controls (Fig. 5B). Curcin treated HCN-1A and MCF-7 showed nearly 50% decrease in FAK expression when compared to control groups. Glioma and HuVEC showed nearly 40% reduced expression of FAK, whereas, L929 and MDA-MB-453 showed very slight decrease in FAK expression. The data clearly implied a significant decrease of FAK levels on curcin treatment, which could have led to the stearic hindrance and physical deformation of actin network.
Vinculin
Another important observation was made with vinculin, which is one of the membrane proteins that play a major role in cell-cell and cell-matrix interaction. The expression of vinculin is essential for the cell to establish contact with its microenvironment. Vinculin expression was completely down-regulated after curcin administration and the cells exhibited loss of structural and mechanical integrity (Fig. 6A). Control group presented vinculin expression throughout the cellular span (Fig. 6b, f, j, n, r, v) whereas the curcin treated cells exhibited diminished expression with lack of discrete signals (Fig. 6d, h, l, p, t, x) indicating the loss of cell's structural integrity, culminating in reduced inter-cellular and cell-substratum contact signaling.
Survivin
Because survivin has a recognized role as an inhibitor of apoptosis, we next investigated whether and how the observed down-regulation of survivin by the toxin would relate to the known ability of curcin to induce apoptosis. Control group of cells presented orderly distributed survivin expression (Fig. 7b, f, j, n, r, v) whereas in the case of curcin treated cells, the expression was significantly arrested (Fig. 7d, h, l, p, t, x) indicating the loss of cell's survival cues and increasing their susceptibility to apoptosis. Thus, the magnitude of survivin down-regulation caused by curcin closely correlated with the extent of apoptosis and with the degree of short-term growth and survival (as determined by alamar blue assay), as well as long-term survival (as determined by 3D cancer colony assay) of these cells.
Lipid peroxidation assay
Mitochondria are known as the main source for the generation of ROS. These ROS are quenched by certain classes of enzymes as superoxide dismutases (SOD) and catalases in the cell. Erythrocytes on the other hand, are incapable of producing SOD and catalases, hence are vulnerable to the extraneous toxicants and the cell membrane may be easily damaged during lipid peroxidation. Lipid peroxides, reactive products of lipid peroxidation, have deleterious effects on the cell membrane. Curcin exposure increased the LPO rate nearly 2 folds when compared to control sample (Fig. 8C). This clearly indicated the ability of curcin to oxidize lipids of cell membrane causing a permanent damage. The cellular ROS generation induced as a response to curcin exposure (Fig. 4A) has already been established, therefore the current observations may be a linked phenomenon.
Hemocompatibility
With the cytotoxic behavior of curcin, one can expect that it exhibits lesser compatibility to blood cells. However, curcin was found to be comparatively lesser toxic towards blood cells. Erythrocyte sedimentation level (Fig. 8A) and hemolysis (Fig. 8B) analysis depicted slight change in the values compared to the control group. The platelet adhesion quantification assay by LDH (Fig. 8D) showed that the adhesion onto curcin-coated plates was extremely lower than on glass or polystyrene substrates proving the hemocompatible nature of curcin. This clearly depicted that curcin is incapable of inducing platelet adhesion and subsequent activation. As platelet activation can lead to complications as primary hemostasis, the inability of curcin to do so could favor in its consideration as a blood-compatible molecule. The thrombus (Fig. 8E) and plasma recalcification profile (Fig. 8F) showed that there is no significant variation of coagulation levels from control samples, though a slight increase in plasma recalcification was found in curcin treated samples post 30 mins, when compared to control samples. The initial slope of the linear portion of each profile was calculated as a measure of the rate of clot formation. No difference was observed between the slopes of control (0.0061) and curcin (0.0055) treated PPP that confirms that curcin is comparatively less coagulative. Though the basic study of hemocompatibility depicted curcin as safer, it is necessary for much detailed study as this knowledge could suggest significant clinical interest.
Discussion
More than 30% of the pharmaceutical agents currently available are of natural origins or their derivatives which form the major source of therapeutic options against a wide range of diseases31,32, signifying not only the advantages of such compounds, but urging the need for newer and more potent versions. In this study, we reported on the multi-dimensional cytocidal effects of curcin on different cell lineages. Previous studies on curcin have shown that like other RIPs, curcin induces cell death33,34 predominantly by inhibiting cell-free translation and protein synthesis in reticulocyte lysate systems35,36. Type 1 RIPs have been portrayed as non-toxic or comparatively lesser toxic agents with respect to the type 2 RIPs as the former are devoid of cell binding moieties which eventually render them incapable of cellular entry on their own, though with exceptions of some specialized cells presenting themselves as vulnerable. Though earlier works narrate the anti-tumor ability of curcin, detailed study on the factors influencing apoptosis have not been reported. We demonstrate the toxic potential of such a type 1 RIP on normal and cancer cell lines alike, which depicts the varying degree of toxicity to the cell types. Curcin significantly inhibited cell proliferation that arrested or degenerated various essential organelles involved in major metabolic pathways. All the cell types, irrespective of normal or cancer origin were susceptible to curcin.
Programmed cell death (PCD), is a genetically regulated process and is pivotal to the homeostasis of all cells. An apoptotic event leading to monitored cellular disorganization culminates from the disturbance of nuclear dynamics complemented by the distortions in the vital cytoplasmic and skeletal organelle structure, function and distribution. It is characterized by cytoplasmic retraction, cytoskeletal disorganization, chromatin condensation, chromosomal DNA fragmentation, mitochondrial swelling with alterations in the membrane potential and permeability, decrease in the anti- and increase in pro-apoptotic stimulus, loss of plasma membrane integrity, decreased metabolic activity and the packaging of cellular constituents into apoptotic vesicles37,38.
Here, we demonstrated that curcin-treated cell lines exhibited several morphological alterations similar to autophagy and apoptosis, predominantly. The cells exhibited enhanced ROS production and mitochondrial damage, with ROS playing additional role in the lipid peroxidation of erythrocytes. Loss of membrane integrity, consequentially the mitochondrial potential, elevated the NF-κB and decreased the anti-apoptotic survivin expression. Survivin's function is to preserve the mitotic apparatus and to allow normal mitotic progression, whereas its anti-apoptotic function is executed via its ability to prevent caspase activation. It has been shown in several other experimental systems that the down-regulation of survivin expression, for example by antisense or siRNA approaches results in elevated “basal level” apoptosis39,40,41. With curcin exposure, mitochondrial membrane potential is lost, leading to the possible release of caspases. Evidently, with the downregulation of survivin, caspase activation is unhindered, leading to the initiation of the apoptotic protocol.
The mitochondrial pore transition is a resultant of increased ROS load in the cytosol, which acts as pro-apoptotic factor influencing the mitochondrial permeability. Mitochondria are crucial in the control of apoptosis, being involved not only in the intrinsic but also the extrinsic pathways. Mitochondrial membrane potential loss is linked to release of apoptotic factors such as cytochrome c and caspases, which in turn activate the downstream apoptotic signaling, guiding the cells to PCD42,43. This mitochondrial pore transition occurs as the secondary effects of ROS elevation in cytosol, attributed here, to curcin exposure. Furthermore, the condensation and shrinkage of nuclear material among treated cell lines, along with PARP inhibition ability of curcin reinforced the notion of an autophagic cell death phenotype. NF-κB is considered an important protein in regulating cellular responses as it belongs to the category of “rapid-acting” primary transcription factors present in cells in an inactive state which do not require new protein synthesis for activation, allowing for a rapid response to harmful cellular stimuli. Interestingly, NF-κB has been described to be activated upon oxidative stress induced by cytotoxic or foreign substances. Upon ROS induction, IκB kinases phosphorylate and trigger degradation of inhibitory IκB proteins. This releases NF-κB resulting in its activation and translocation into nucleus and act as transcription factor for apoptosis44,45,46. The clear correlation of NF-κB activation with ROS levels suggests that NF-κB activation occurs under oxidative stress. We attribute the observed nuclear toxicity by curcin to be from the translocation of NF-κB from cytoplasm to nucleus, triggering the apoptosis-signaling pathway. In addition, PARP1 is known to be overexpressed in variety of cancers and its expression has been associated with overall prognosis in cancer, especially breast cancer47. A series of new therapeutic agents that are potent inhibitors of the PARP1 and PARP2 isoforms have demonstrated important clinical activity in patients with cancers48. The loss of base excision repair capacity produced on PARP inhibition by curcin can prompt the evaluation of this RIP as potential enhancer of DNA damaging cytotoxic chemotherapeutic molecules such as alkylating agents (for example, platinum, cyclophosphamide) and topoisomerase 1 inhibitors.
The morphological changes observed in curcin treated cells were in accord with previous observations and presented ultrastructural alterations of organelles. It is widely known that apoptotic cells reorganize their cytoskeleton, with fragmentation of the microfilament bundles conferring plasticity to the cell and promote volume decrease as well as cell shrinkage49. On the other hand, cytoskeletons are also important in the execution phase, when they form a cortical network under the plasmalemma, thus preserving cell morphology and plasma membrane integrity. The maintenance of these structures depends on high cellular ATP levels, so that they undergo disorganization in late apoptosis, when mitochondria suffer extensive depolarization. Using drugs that influence actin stabilization/depolymerization, it has been shown that stimulating alterations in the microfilaments status is sufficient to induce apoptosis50. Curcin treated cell lines also exhibited a considerable retraction of the cellular mass with detrimental effects on the cytoskeletal proteins, vinculin and actins. Earlier reports show that vinculin promotes traction force at FA, mediating conversion of forces generated in the cytoskeleton that drive retrograde flow into traction force on the ECM during FA maturation. The tail domain of Vinculin associates with F-actin acting as an effective component of the molecular clutch in determining the architecture of cell to matrix attachment. FAK is a nonreceptor protein tyrosine kinase that predominantly localizes in focal adhesion complexes (FAC). The actin remodeling or disruptions break up the FACs, thereby drastically affecting any activation of FAK, which is functionally linked to mature and stable FACs. The reduced cytoskeletal remodeling is thus likely to enlarge the initial cytoskeletal aberrations induced directly by curcin. As FACs are multiprotein complexes and form direct contact between actin and extracellular environment, they have a role in actin mediated signaling51,52. Thus, the loss of mature FACs on curcin treatment implies significant aberrations in intracellular signaling pathways, which could culminate in a diverse variety of malfunctions in cellular homeostasis and should be considered as a highly warranted and extremely significant toxicological effect of the RIP. It is also worth noting that the different organelles have reciprocal interactions with each other, both in viable and apoptotic cells. The actin cytoskeleton and microtubules can also interact with mitochondria, which are considered as the central processing organelles in most apoptotic pathways. Apoptotic signals (either from outside or inside the cell) converge to mitochondria that can initiate the execution phase of apoptosis.
We also observed lower metabolic activity and 3D tumor suppression post curcin exposure. Apart from the prominent apoptotic indications, cyto-necrosis due to curcin was observed which is thought to occur via binding of the protein to cell membrane, causing membrane destabilization, facilitating easy entry of propidium iodide.
Drugs intended for long-term contact with blood must be subjected to thorough scrutiny, since hemocompatibility is the most important aspect during any drug administration. Prospective drug candidates in contact with blood must not induce any pernicious effects as thrombosis, thromboembolisms, antigenic responses, destruction of blood constituents and plasma proteins, etc. In this study, the hemocompatibility of curcin was investigated by analyzing the hemolytic index, erythrocyte sedimentation rate, plasma recalcification and platelet adhesion. Hemolysis was less than 5% for test samples and was well within the permissible limit set for biomaterials. ESR rate also supports the claim that curcin exposure did not cause significant deleterious effects. In addition, the platelet adhesion study as well as plasma recalcification analysis assures the blood compatible nature of curcin. Being an RIP, the observations made were interesting in lieu of considering curcin for further in vivo therapeutic analysis.
In conclusion, we project curcin as a highly cogent toxic RIP capable of multiple modules of action on shutting down the cellular machinery. The multitudes of activities encompassing ROS generation, loss of cellular organelle function and integrity, decline of mitochondrial membrane potential, induction of NF-κB, disruption of the cytoskeletal scaffold leading to cellular collapse, suppression of FAK and more interestingly the downregulation of survivin and membrane adhesion proteins, in all culminating into PCD. The 3D cancer mass regression and complete disruption by curcin provides concrete supplementary proof for its inevitable activity on malignant solid tumors, eliminating the core of the cancer mass and with it, the chance of future proliferation and recurrence. As many RIPs have been proposed and tested as potent immunotoxins and few in nano-drug delivery for specific tumor targeting and on the basis of the comprehensive destruction of cancer cells in nearly every possible aspect, we present curcin as a potent option not only as a robust cytocidal phytomolecule, but also as a puissant means to curb the progression of cancer-metastasis.
Methods
Cell culture
L929 (mouse fibroblast), MCF-7 and MDA-MB-453 (breast cancer), Glioma (brain cancer) were from Riken Bioresources, Japan and HCN-1A (neuronal) was from ATCC. HuVEC was from Gibco. L929, HCN-1A, MCF-7 and Glioma were maintained in T25 flasks using DMEM medium supplemented with FBS, while HuVEC was grown in Med 200 supplemented with LSGS and antibiotics (100 U/ml penicillin and streptomycin) and incubated in a 5% CO2 incubator at 37°C. MDA-MB-453 were maintained in L-15 media supplemented with 10% FBS in an incubator at 37°C. The cells were sub-cultured every two-three days. For all confocal experiments, cells were cultured in 35 mm glass base dishes (Iwaki) 24 h.
Cytotoxicity assay
Approximately 5000 cells were seeded into each well of 96 well plates. After attaining visual confluency, curcin was added at different concentrations (100 μg/ml, 10 μg/ml and 1 μg/ml). Control group was devoid of curcin. After 72 h of incubation with curcin, cells were washed with PBS and 0.2 ml of respective medium was added. Cell viability was assayed with alamar blue in a 96 well plate reader (Multidetection microplate scanner, Dainippon Sumitomo Pharma).
Alamar blue assisted visual fluorescence detection of cytotoxicity
Curcin (100 μg/ml) was added to the test cells and incubated for 72 h. Cells were washed thoroughly with PBS and observed in PBS at an excitation wavelength of 561 nm in a confocal laser scanning microscope (CLSM, Olympus IX 81 under DU897 mode).
Analysis of apoptosis and necrosis
Apoptotic and necrotic dead cell staining was performed employing annexin-V and PPI respectively. The test cells were treated with curcin at 100 μg/ml for 72 h. Cells were washed post treatment, stained and imaged using CLSM under 488 and 561 nm excitation.
ROS generation
Cells were cultured in 96 well plates, 5000 cells/well and treated with 100 μg/ml, 10 μg/ml and 1 μg/ml of curcin. Control groups and blanks were maintained. After 72 h of treatment, 20 μl of DCFH-DA dye was added to each well at a concentration of 10 mM. The plate was incubated for 30 min and the fluorescence in each well was quantified at 480 nm excitation using a microplate reader. The fluorescence of the test and control cells was directly proportional to the amount of ROS produced.
NF-κB and FAK estimation
Approximately 5000 cells were seeded onto 96 well plates. Cells were grown under ambient condition for 24 h. 100 μg/ml of curcin was added to test wells and the plates were maintained at culture conditions for 72 h. The NF-κB estimation was carried out as per manufacturer's instruction and resultant absorbance was read spectrophotometrically at 490 nm. FAK estimation was carried out as per manufacturer's instruction with cell maintenance and curcin treatment conditions similar to NF-κB estimation.
Mitochondrial membrane transition pore assay
Approximately 25,000 cells were seeded onto 35 mm glass base dishes. Test group of cells were treated with 100 μg/ml of curcin. After 72 h, the plates were stained for pore transition as per manufacturer's instruction and analyzed.
PARP inhibition assay
100, 10 or 1 μg/ml of curcin was incubated along with PARP enzyme and PARP cocktail mix, followed by assessing the inhibition rate of PARP according to the manufacturer's instructions. Negative control was maintained without curcin exposure.
Organelle disruption study
Curcin (100 μg/ml) was added to the test cells and incubated for 72 h with observations being made every 24 h till 72 h. DAPI and Mitotracker were freshly prepared and added to the cells. After incubation of 10 min (DAPI) and 45 min (Mitotracker) cells were washed with PBS (pH 7.4) and fresh media added prior to observation. Actin cytoskeletal staining was performed with phalloidin rhodamine on the 3rd day of curcin treatment. Cells were visualized at an excitation wavelength of 405 nm (DAPI), 488 nm (Mitotracker) and at 561 nm (Phalloidin Rhodamine).
Immunocytochemical assays for vinculin and survivin
Cells were treated with 100 μg/ml concentration of curcin for 72 h. After the incubation period, cells were washed and fixed using formaldehyde and acetone and treated with antibodies to either survivin or vinculin. After 1 h, the secondary fluorescent antibodies (Goat polyclonal Secondary Antibody to Mouse IgG - H&L (FITC) abcam) were added and the plates were incubated for 1 h. Plates were then washed and observed under CLSM.
3D cancer colony assay
Glioma, MCF-7 and MDA-MB-453 were loaded onto thick (3 mm) Geltrex® (Invitrogen) basement membrane matrix coated 96 well plates. The well-established cancer colonies (post 96 h) were supplemented with 100 μg/ml of curcin and effects monitored periodically. Live dead staining was performed using Calcein/PPI according to manufacturer's instructions, post 96 h curcin treatment.
Lipid peroxidation (LPO) assay
The reaction of MDA with TBA has been widely adopted as a sensitive assay method for lipid peroxidation53. LPO assay characterizes the degradation of the TBA and the concentration of melondialdehyde reacted in vitro to give a fluorescent product that can be measured at 532 nm. Lipid peroxidation in RBCs due to curcin exposure was analyzed accordingly. 0.2 ml of healthy human blood (collected from volunteer) was incubated with 1.5 mL thiobarbituric acid, 0.2 mL of SDS and 1.5 mL of 20% acetic acid solution at 100°C for 1 h. Post incubation, the solution was centrifuged at 3500 rpm for 10 min and the supernatant was collected to measure the OD value.
Hemolysis
100 μl of blood was incubated with 100 μl (100 μg/ml) of curcin solution for 3 h at 37°C. Positive and negative controls were maintained employing the use of either 100 μl of Triton-X and 40% PEG respectively. The mixtures were centrifuged at 800 g for 15 min. The degree of hemolysis was determined by measuring the absorbance of the supernatant at 540 nm, as previously reported54. The absorbance value accounted for the amount of hemoglobin release induced by curcin.
Erythrocyte sedimentation
100 μl of erythrocyte suspension (1 × 106 cells/ml) was incubated with 100 μl (100 μg/ml) of curcin or 40% PEG (negative control) or with Triton-X (positive control) in eppendorf for 1 h at 37°C. The erythrocyte sedimentation was investigated and recorded using digital camera.
Whole blood clotting time (thrombus formation)
The thrombogenicity of curcin was evaluated using a whole blood kinetic clotting time method. Briefly, the clotting reaction was activated with the addition of 100 μl of (100 μg/ml) curcin to l00 μl of blood sample and incubated at 37°C for 15 min. Control samples are maintained without curcin exposure. Post incubation, to 50 μl of the test mixture, 25 μl of CaCl2 was added. All samples were incubated at room temperature for 5, 20, 40 and 60 min. At the end of each time point, the samples were incubated with 3 ml of distilled water for 5 min. Each well was sampled in triplicate (200 μl each) and transferred to a 96-well plate. The red blood cells that were not trapped in a thrombus were lysed with the addition of distilled water, thereby releasing hemoglobin into the water for subsequent measurement. The concentration of hemoglobin in solution was assessed by measuring the absorbance at 540 nm using a 96 well plate reader. The size of the clot is inversely proportional to the absorbance value.
Platelet adhesion
Platelet rich plasma (PRP) were isolated as previously described55. PRP was dropped onto bare polystyrene culture plates or curcin coated culture plates and was incubated for 1 h at 37°C under static conditions. The suspension was aspirated and each well was rinsed carefully three times with PBS. The number of adherent platelets was determined by detecting the amount of lactate dehydrogenase (LDH) present after cell lysis. Briefly for LDH analysis, adherent platelets were lysed by incubation with 2% Triton-PBS buffer for 30 min at 37°C. A colorimetric substrate for LDH was added and incubated for 20 min at 37°C. The reaction was stopped and the optical density was measured at 490 nm with reference wavelength of 650 nm.
Plasma recalcification profile
Platelet poor plasma (PPP) was utilized for this study and was prepared as previously described56,57. 100 μl of test sample were incubated with 100 μl of PPP in 96 well plates. Controls consisted of tissue culture-treated plastic (TCP) exposed to PPP with and without CaCl2. Following the addition of PPP, 100 μl of 0.025 M CaCl2 was added to each well (except without Ca+2, negative control). The plate was then immediately placed in a 96 well plate reader, where the kinetics of the clotting process due to recalicification were monitored by measuring the absorbance at 405 nm (every 3 min for 45 min) at 37°C. In calculating the mean absorbance at each time point, three wells were averaged per sample. The slope of the linear portion of each profile were calculated and analyzed.
Statistical analysis
All quantitative experiments were conducted with at least three independent experiments. Statistical evaluation was performed using Origin Pro Software. Data were analyzed by a one-way ANOVA for multiple data set analyses and were expressed as mean ± SD. A difference was considered statistically significant when p < 0.05.
References
Stirpe, F., Barbieri, L., Battelli, M. G., Soria, M. & Lappi, D. A. Ribosome–Inactivating Proteins from Plants: Present Status and Future Prospects. Nat. Biotechnol. 10, 405–412 (1992).
Stripe, F. Ribosome-inactivating proteins: From toxins to useful proteins. Toxicon 67, 12–16 (2013).
Barbieri, L., Battelli, M. & Stripe, F. Ribosome-inactivating proteins from plants. Biochim. Biophys. Acta 1154, 237–282 (1993).
Endo, Y., Mitsui, K., Motizuki, M. & Tsurugi, K. The mechanism of action of ricin and related toxic lectins on eukaryotic ribosomes. J. Biol. Chem. 262, 5908–5912 (1978).
Endo, Y. & Tsurugi, K. RNA N-glycosidase activity of ricin A-chain, mechanism of action of the toxic lectin on eukaryotic ribosomes. J. Biol. Chem. 262, 8128–8130 (1987).
Stripe, F. Ribosome-inactivating proteins. Toxicon 44, 371–383 (2004).
Barbieri, L., Ferreras, J. M., Barraco, A., Ricci, P. & Stirpe, F. Some ribosome-inactivating proteins depurinate ribosomal RNA at multiple sites. Biochem. J. 286, 1–4 (1992).
Stirpe, F. & Battelli, M. G. Ribosome-inactivating proteins: progress and problems. Cell. Mol. Life Sci. 63, 1850–1866 (2006).
Ng, T. B., Chan, W. Y. & Yeung, H. W. Proteins with abortifacient, ribosome inactivating, immunomodulatory, antitumor and anti-AIDS activities from cucurbitaceae plants. Gen. Pharmac. 23, 575–590 (1992).
Lin, J. Y., Tserng, K. Y., Chen, C. C., Lin, L. T. & Tung, T. C. Abrin and ricin: new anti-tumour substances. Nature 227, 292–293 (1970).
Kanellos, J., McKenzie, I. F. & Pietersz, G. A. Intratumour therapy of solid tumours with ricin-antibody conjugates. Immunol. Cell Biol. 67, 89–99 (1989).
Ramakrishnan, S., Fryxell, D., Mohanraj, D., Olson, M. & Li, B. Cytotoxic conjugates containing translational inhibitory proteins. Ann. Rev. Pharmacol. Toxicol. 32, 579–621 (1992).
Xia, H. C., Li, F., Li, Z. & Zhang, Z. C. Purification and characterization of Moschatin, a novel type I ribosome-inactivating protein from the mature seeds of pumpkin (Cucurbita moschata) and preparation of its immunotoxin against human melanoma cells. Cell Research 13, 369–374 (2003).
Daniels1, T. R. et al. Conjugation of an anti–transferrin receptor IgG3-avidin fusion protein with biotinylated saporin results in significant enhancement of its cytotoxicity against malignant hematopoietic cells. Mol. Cancer Ther. 6, 2995–3008 (2007).
Pirie, C. M., Liu, D. V. & Wittrup, K. D. Targeted cytolysins synergistically potentiate cytoplasmic delivery of gelonin immunotoxin. Mol. Cancer Ther. 12, 1774–82 (2013).
Ghetie, M. A. & Vitetta, E. S. Recent developments in immunotoxin therapy. Curr. Opin. Immunol. 6, 707–714 (1994).
Narayanan, S., Surendranath, K., Bora, N., Surolia, A. & Karande, A. A. Ribosome inactivating proteins and apoptosis. FEBS Letters 579, 1324–1331 (2005).
Bolognesi, A. et al. Induction of apoptosis by ribosome-inactivating proteins and related immunotoxins. Int. J. Cancer 68, 349–355 (1996).
Sikriwal, D., Ghosh, P. & Batra, J. K. Ribosome inactivating protein saporin induces apoptosis through mitochondrial cascade, independent of translation inhibition. Int. J. Biochem. Cell Biol. 40, 2880–2888 (2008).
Cheung, M. C. et al. An evolved ribosome-inactivating protein targets and kills human melanoma cells in vitro and in vivo. Molecular Cancer 9, 28 (2010).
Xiong, S. D. et al. Ribosome-inactivating proteins isolated from dietary bitter melon induce apoptosis and inhibit histone deacetylase-1 selectively in premalignant and malignant prostate cancer cells. Int. J. Cancer 125, 774–782 (2009).
Thomas, R., Sah, N. K. & Sharma, P. B. Therapeutic biology of Jatropha curcas: a mini review. Curr. Pharm. Biotechnol. 9, 315–24 (2008).
Pandey, V. C. et al. Jatropha curcas: A potential biofuel plant for sustainable environmental development. Renewable and Sustainable Energy Reviews 16, 2870–2883 (2012).
Debnath, M. & Bisen, P. S. Jatropha curcas L., a multipurpose stress resistant plant with a potential for ethnomedicine and renewable energy. Curr. Pharm. Biotechnol. 9, 288–306 (2008).
Felke, J. The poisonous principles of the seeds of Jatropha curcas Linn. Landw. Versuchsw. 82, 427–430 (1914).
Juan, L., Fang, Y., Lin, T. & Fang, C. Antitumor effects of curcin from seeds of Jatropha curcas. Acta Pharmacol. Sin. 24, 241–246 (2003).
Lin, J. et al. Cloning and expression of curcin, a ribosome-inactivating protein from the seeds of Jatropha curcas. Acta Bot. Sin. 45, 858–863 (2003).
Qin, W., Ming-Xing, H., Ying, X., Xin-Shen, Z. & Fang, C. Expression of a ribosome inactivating protein (curcin 2) in Jatropha curcas is induced by stress. J. Biosci. 30, 351–357 (2005).
Zheng, Q. et al. Expression of curcin-transferrin receptor binding peptide fusion protein and its anti-tumor activity. Protein Expr Purif. 89, 181–188 (2013).
Mohamed, M. S. et al. Type 1 ribotoxin-curcin conjugated biogenic gold nanoparticles for a multimodal therapeutic approach towards brain cancer. Biochim Biophys Acta. (2013) http://dx.doi.org/10.1016/j.bbagen.2013.12.020.
Mishra, B. B. & Tiwari, V. K. Natural products: an evolving role in future drug discovery. Eur. J. Med. Chem. 46, 4769–807 (2011).
Mondal, S. et al. Natural products: promising resources for cancer drug discovery. Anticancer Agents Med Chem. 12, 49–75 (2012).
Luo, M. J. et al. Expression, purification and anti-tumor activity of curcin. Acta Biochim. Biophys. Sin. (Shanghai). 38, 663–668 (2006).
Zhao, Q., Wang, W., Wang, Y., Xu, Y. & Chen, F. The effect of curcin from Jatropha curcas on apoptosis of mouse sarcoma-180 cells. Fitoterapia 83, 849–852 (2012).
Lin, J., Yan, F., Tang, L. & Chen, F. Antitumor effects of curcin from seeds of Jatropha curcas. Acta Pharmacol. Sin. 24, 241–246 (2003).
Zheng, Q. et al. Expression of curcin-transferrin receptor binding peptide fusion protein and its anti-tumor activity. Protein Expr. Purif. 89, 181–188 (2013).
Kroemer, G. et al. Classification of cell death: recommendations of the nomenclature committee on cell death. Cell. Death Differ. 16, 3–11 (2009).
Guimarães, C. A. & Linden, R. Programmed cell deaths. Apoptosis and alternative deathstyles. Eur. J. Biochem. 271, 1638–1650 (2004).
Pyrko, P. et al. Downregulation of survivin expression and concomitant induction of apoptosis by celecoxib and its non-cyclooxygenase-2-inhibitory analog, dimethyl-celecoxib (DMC), in tumor cells in vitro and in vivo. Mol. Cancer 5, 19 (2006).
Altieri, D. C. et al. Survivin, versatile modulation of cell division and apoptosis in cancer. Oncogene, 22, 8581–8589 (2003).
Pennati, M. et al. Ribozyme-mediated down-regula- tion of survivin expression sensitizes human melanoma cells to topotecan in vitro and in vivo. Carcinogenesis 25, 1129–1136 (2004).
Green, D. R. & Kroemer, G. The pathophysiology of mitochondrial cell death. Science 305, 626–629 (2004).
Bottone, M. G. et al. Morphological Features of Organelles during Apoptosis: An Overview. Cells 2, 294–305 (2013).
Morgan, M. J. & Liu, Z. Crosstalk of reactive oxygen species and NF-κB signaling. Cell Research 21, 103–115 (2011).
Bae, Y. S., Oh, H., Rhee, S. G. & Do, Y. Y. Regulation of reactive oxygen species generation incell signaling. Molecules and Cells 32, 491–509 (2011).
Wan, F. & Lenardo, M. J. The nuclear signaling of NF-kappaB: current knowledge, new insights and future perspectives. Cell Research 20, 24–33 (2010).
Rojo, F. et al. Nuclear PARP-1 protein overexpression is associated with poor overall survival in early breast cancer. Ann. Oncol. 23, 1156–1164 (2012).
Kummar, S. et al. Advances in using PARP inhibitors to treat cancer. BMC Med. 10, 25 (2012).
Núñez, R., Sancho-Martínez, S. M., Novoa, J. M. & López-Hernández, F. J. Apoptotic volume decrease as a geometric determinant for cell dismantling into apoptotic bodies. Cell Death Differ. 17, 1665–1671 (2010).
Ono, S. Mechanism of depolymerization and severing of actin filaments and its significance in cytoskeletal dynamics. Int. Rev. Cytol. 258, 1–82 (2007).
Grashoff, C. Measuring mechanical tension across vinculin reveals regula- tion of focal adhesion dynamics. Nature 466, 263–266 (2010).
Thievessen, I. et al. Vinculin-actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth. J Cell Biol. 202, 163–177 (2013).
Scott, B. C. et al. Lipoic and dihydrolipoic acids as antioxidants. A critical evaluation. Free Radic. Res. 20, 119–133 (1994).
Katsu, T., Ninomiya, C. & Kuroko, M. Action mechanism of amphipathic peptides gramicidin S and melittin on erythrocyte membrane. Biochim. Biophys. Acta 939, 57–63 (1988).
Grunkemeier, J. M., Tsai, W. B. & Horbett, T. A. Hemocompatibility of treated polystyrene substrates: contact activation, platelet adhesion and procoagulant activity of adherent platelets. J. Biomed. Mater. Res. 41, 657–670 (1998).
Motlagh, D., Yang, J., Lui, K. Y., Webb, A. R. & Ameer, G. A. Hemocompatibility evaluation of poly(glycerol-sebacate) in vitro for vascular tissue engineering. Biomaterials 24, 4315–4324 (2006).
Tamada, Y., Kulik, E. A. & Ikada, Y. Simple method for platelet counting. Biomaterials 16, 259–261 (1995).
Acknowledgements
M. Sheikh Mohamed would like to thank the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, for financially supporting this research. Part of this study has been supported by a grant for the programme of strategic research foundation at private universities S1101017, organized by the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan since April 2012. We, thank the Japan Bio-Energy development Corporation (JBEDC), Japan, for generously providing the seeds of Jatropha curcas for this study.
Author information
Authors and Affiliations
Contributions
M.S.M., S.V. and D.S.K. conceived and designed the experiments and drafted the final manuscript. H.M., Y.S.1 and A.E. assisted in chromatography and electrophoresis. Y.N. assisted in CLSM studies. A.A. assisted in flow cytometry. Y.S.2 contributed in HPLC and blood compatibility analysis. A.C.P. assisted in statistical analysis. Y.Y., T.M. and D.S.K. analyzed the data and contributed reagents/materials/analysis tools. All authors read and approved the manuscript.
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Electronic supplementary material
Supplementary Information
Supplementary data
Rights and permissions
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder in order to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
About this article
Cite this article
Mohamed, M., Veeranarayanan, S., Minegishi, H. et al. Cytological and Subcellular Response of Cells Exposed to the Type-1 RIP Curcin and its Hemocompatibility Analysis. Sci Rep 4, 5747 (2014). https://doi.org/10.1038/srep05747
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/srep05747
- Springer Nature Limited
This article is cited by
-
Anodically Grown Titania Nanotube Induced Cytotoxicity has Genotoxic Origins
Scientific Reports (2017)