In vitro antitumor, pro-inflammatory, and pro-coagulant activities of Megalopyge opercularis J.E. Smith hemolymph and spine venom
Ethical statement
All methods involving human samples were performed in accordance with Institutional guidelines and regulations. Volunteers donating blood samples for experiments in this study provided a signed informed consent and remained anonymous. The donor sample consent informs and the assay involving human samples were reviewed and approved by the Institutional Ethics Committee at Autonomous University of Nuevo Leon (UANL). Experiments related to the use of animals were reviewed and approved by the Institutional Committee for Research Ethics and Animal Welfare of “The College of Biological Sciences” (CEIBA) at UANL with application number CEIBA-2017-005, following Mexican regulation NOM-062-ZOO-1999 entitled Technical Specifications for the Production, Care and Use of Laboratory Animals, normative that aligns with the guidelines and basic principles in the NIH Guide for the Care and Use of Laboratory Animals. In addition, standard ethical guidelines for ascites tumor induction in mice and rats36 were followed for experiments involving tumor cells obtained from tumor-bearing mice.
Reagents, culture media, and tumor cell line
Penicillin–Streptomycin solution, and RPMI 1640 and AIM-V media were obtained from Life Technologies (Grand Island, NY). Fetal bovine serum (FBS), Actinomycin D, dimethyl sulfoxide (DMSO), and 3-[4,5-dimethyl thiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were purchased from Sigma-Aldrich (St. Louis, MO). Taq & Go Master Mix 5X, pGEM-T Easy plasmid, and all molecular biology reagents were obtained from Promega (Madison, WI). Oligonucleotides were synthesized by Integrated DNA Technologies (UNIPARTS S.A., Monterrey, N.L., Mexico).
The tumor cell line L5178Y-R (mouse DBA/2 lymphoma) was obtained from The American Type Culture Collection (Rockville, MD), and maintained in culture flasks with RPMI 1640 medium supplemented with 10% FBS, 1% L-glutamine, and 0.5% Penicillin–Streptomycin solution (referred as complete RPMI 1640 medium) at 37 ºC, in a humidified atmosphere of 5% CO2 in air. Cellular density was kept between 105 and 106 cells/mL.
Animals and tumor intraperitoneal implantation
Six- to eight-week old BALB/c female mice were purchased from Harlan Mexico S.A. de C.V. (Mexico, D.F.). Regarding housing conditions, up to five animals per cage were kept in a pathogen- and stress-reduced environment at 24 °C, under a light–dark cycle (light phase, 06:00–18:00 h) in a One Cage 2100 System (Lab Products, Inc., Seaford, DE) and given water and food ad libitum36. Three mice were used for L5178Y-R lymphoma induction, which was performed by intraperitoneal (i.p.) administration of 0.2 mL of L5178Y-R tumor cells suspension (5 × 106 cells/mouse). After 13 d inoculation, mice were euthanized by cervical dislocation and peritoneal cavity ascites was collected. The ascites suspension was placed in a 50 mL tube containing 10 mL PBS for in vitro cytotoxicity assays37.
Insect source and rearing conditions
Venomous caterpillars were collected from the escarpment live oak Quercus virginiana var. fusiformis Mill. (Fagaceae) trees growing in the Cumbres National Park of Sierra Madre Oriental, in Monterrey, Nuevo Leon, located northeastern México at 25° 42′ 28.8″ N and 100° 22′ 11.4″ W. Insects collection was performed with a collaboration of Biological Science College (UANL) and the Environmental Education Program of the Wild-Life Cumbres National Park of Nuevo Leon State (Parques y Vida Silvestre, https://www.nl.gob.mx/servicios/programa-de-educacion-ambiental). Collected larvae and escarpment live oak leaves were placed inside of a 2-L glass jar with a 2 cm × 2 cm open square metallic cap, covered with wire mesh screen for air exchange. Collected material was transported to the laboratory for larval rearing. Jars with larvae and leaves were incubated at 25 ± 2 °C, 65% ± 5% relative humidity, and 16:8 h light:darkness cycles, inside of a rearing insect room. Larvae were fed on fresh escarpment live oak leaves, previously rinsed in tap water for 30 s. Incubated larvae were tested after reaching the fourth instar. Extra reared larvae were kept feeding until reaching the pupa stage, followed by adults’ emergence, in order to generate and maintain new insect colonies for further experiments.
Caterpillar venom molecular identification
DNA from three fourth instar caterpillar larvae was extracted, using the Wizard Genomic DNA Purification Kit (Promega) and following the isolating genomic DNA from tissue culture cells and animal tissue protocol. DNA extract was used as a template for PCR amplification of specific primers for the cytochrome oxidase subunit (COI) F1 5′AAC WYT ATA YTT TAT TTT TGG 3′ R and 5′TGT TGR TAW ARR ATW GGR TC 3′, designed from Genbank Megalopyge genus sequences.
PCR was performed using GoTaq Green Master Mix (Promega) in a 50 µL volume, with 100 ng of DNA as template and 1 µM of forward and reverse primers. Thermal cycling conditions included an initial denaturation step at 94 °C for 10 min, followed by 35 cycles of denaturation at 94 °C for 40 s, annealing at 60 °C for 40 s, and elongation at 72 °C for 2 min.
Amplified PCR products were ligated into pGEM-T Easy (Promega) in competent E. coli TOP-10 cells. Detected plasmids were purified using the Wizard Plus SV Minipreps DNA Purification System. Sanger sequencing was performed with standard vector M13F and M13R primers by the Instituto de Biotecnología at Universidad Nacional Autónoma de México. The sequence obtained was analyzed on platform Boldsystem.
HEV and SSV spine setae samples
HEV was obtained by performing a puncture on the third false leg from each larva head. Released fluid (~ 200 µL) was collected and centrifuged at 9,600 rpm for 2 min. The resulting supernatant protein content was quantified on a NanoDrop Lite kit and adjusted to 1 mg/mL. This was used as a stock for further dilution and dosage preparations38. In addition, SSV was obtained from four reared fourth instar venomous caterpillars, extracted according to da Silva et al.39. Spine setae were cut from the caterpillars’ integument, homogenized, sonicated in sterile PBS, and processed as described for HEV.
HEV and SSV cytotoxicity against murine L5178Y-R lymphoma cells
To determine the direct in vitro effect of HEV and SSV on tumor cell growth, L5178Y-R cell suspensions (from i.p. lymphoma grown in female BALB/c mice as explained above) were adjusted to 5 × 104 cells/mL in complete RPMI 1640 medium. We evaluated the antitumor effect of a broad range of concentrations of HEV and SSV, following the cytotoxicity assay previously described15. One hundred microliters of the cell suspensions were then added to flat-bottomed 96-well plates (Becton Dickinson, Lincoln Park, NJ), containing triplicate cultures (100 µL) of complete RPMI 1640 medium (unstimulated control), HEV or SSV (7.8–500 µg/mL)37, using 3.1–125 µg/mL Vincristine (Sigma-Aldrich), as positive control. After incubation for 44 h at 37 °C in 5% CO2, MTT (0.5 mg/mL, final concentration) was added, and cultures were incubated for additional 4 h. Cell cultures were then incubated for 16 h with 100 µL DMSO to dissolve formazan crystals, and optical densities (ODs) were read in a microplate reader (Bio-Tek Instruments, Inc., Winooski, VT) at 540 nm37. Percentage of cytotoxicity was calculated as follows:
$$ % {text{ Cytotoxicity}}, = ,{1}00 – left[ {left( {{text{OD}}_{{{54}0}} {text{in HEV{-} or SSV{-}treated cells}}/{text{OD}}_{{{54}0}} {text{in untreated cells}}} right), times ,{1}00} right]. $$
The Statistical Package for the Social Sciences version 17.040, was used to calculate the inhibitory concentration at 95% (IC95), selecting the Probit analysis.
Apoptosis assay
Cellular death type resulting from HEV- or SSV-mediated L5178Y-R cytotoxicity was determined according to Reyna-Martínez et al.41. For this, 3 × 106 cells were exposed to HEV or SSV IC50 using flat-bottomed, 24-well plates (Becton Dickinson), and incubated for 24 h under the same conditions as for the cytotoxicity assay. Treated cells were aliquoted into microtubes, washed by centrifugation at 9,600 rpm (Sorvall ST16R Centrifuge; ThermoScientific, Pittsburgh, PA), and suspended in 500 μL of complete RPMI 1640 medium. Cells were then stained adding 1 μL of 100 μg/mL acridine orange and 1 μL of 100 μg/mL ethidium bromide, and incubated for 5 min. Next, cultured cells were washed three times by centrifugation 9600 rpm with 1 mL PBS and suspended in 100 μL of PBS 1×, after which 10 μL of cell suspension samples were observed in a fluorescence microscope adapted with a rhodamine filter (540–570 nm), using Actinomycin D (800 ng/mL) as positive control.
Uniform green stained cells were quantified as viable cells and spotty green or granular core cells were quantified as in early apoptosis. Orange dots or cells with large granules similar to those observed in early-apoptosis cells were quantified as in late apoptosis, whereas uniform orange hue cells were quantified as in necrosis42.
Staining cells results were validated by the DNA degradation method41, where DNA like-ladder fragmentation indicates apoptotic activity, whereas DNA smear represents cell death by necrosis. DNA extracted from 1 × 106 cells per treatment were tested using the AxyPrep Multisource Genomic DNA Miniprep kit (Axygen) in 1% agarose gel electrophoresis at 100 V for one hour. The gel was then stained with 5 ng/mL ethidium bromide and analyzed on a GelDoc XR photo-documenter (Bio Rad, Berkeley, CA).
Lymphocyte proliferation assay
The effect of venom caterpillar HEV and SSV extracts on murine lymphocyte proliferation was determined by the MTT reduction colorimetric technique37. Two mice were euthanized and thymuses were immediately removed after mice death, a single cell-suspension was prepared by disrupting the organs in RPMI 1640 medium, as previously reported37. Cell suspensions were then washed three times in this medium, suspended, and adjusted to 1 × 107 cells/mL in complete RPMI 1640 medium. One hundred microliters of thymus cell suspensions were added to flat-bottomed 96-well plates (Becton Dickinson) containing triplicate cultures (100 µL) of complete RPMI 1640 medium (unstimulated control), HEV and SSV at 7.8, 15.6, 31.25, 62.5, 125, 250, and 500 µg/mL15,37, and the positive control Concanavalin A (6.25 μg/mL) for 48 h at 37 °C in 95% air-5% CO2 atmosphere. After 44 h of incubation, MTT (0.5 mg/mL, final concentration) was added, and cultures were incubated for additional 4 h. Cell cultures were then incubated for 16 h with 100 µL of DMSO and ODs, resulting from dissolved formazan crystals, were then read in a microplate reader (DTX 880 Multimode detector, Becton Dickinson, Austria) at 570 nm37. To calculate the lymphoproliferation index, the obtained values between the samples were compared. For this, values recorded by extracts treated cells were divided with the value given by Concanavalin A (tested as mouse T-cell mitogen) as follows: OD570 in treated cells/OD570 in Concanavalin A treated cells. Therefore, all values were compared with the control, where the lowest concentrations have a value of 1, since there was no difference compared with the control.
Human peripheral blood mononuclear cells (hPBMC) cytokine response to M. opercularis extracts
Cytokine production by hPBMC was measured after HEV and SSV extracts exposure. For this, hPBMC were isolated with Ficoll-Paque Plus (GE Healthcare, Uppsala, Sweden) and adjusted to 1 × 106 cells/mL in complete RPMI 1640 medium. One hundred microliters of the cell suspension were placed in a 96-well plate in the presence or absence (untreated control) of 100 μL of HEV or SSV M. opercularis extracts at 3.91, 7.81, 15.62, 31.25, 62.5, and 125 µg/mL15 in complete RPMI 1640 medium. Plates were then incubated at 37 °C for 48 h and centrifuged at 400 rpm for 5 min.
Cell‐free supernatants were then subjected to IL-1β, IL-6, IL-8, and TNF-α levels determination by cytometric bead arrays (CBA) (BD Biosciences, San Jose, CA) on a BD Accuri C6 Flow Cytometer Sampler (BD Biosciences, Ann Arbor, MI), following manufacturer’s instructions, and data analyzed with the FCAP Array v3.0 (SoftFlow Inc.). Results were adjusted by subtracting the basal levels of cytokines from untreated hPBMC (negative control) and data analyzed by Prism 6 software (GraphPad Software Inc., La. Jolla, CA)43.
Coagulation assay
The effect of HEV and SSV activity on plasma coagulation was assessed, using the re-calcification time assay44, adapted for a microplate reader. For this, 1 mg/mL HEV and SSV reactive samples were prepared in 20 mM Tris–HCl buffer pH 7.4 and sterilized by filtration with a 0.22 μm micropore filter. Reactive samples consisted of 50 μL of citrated human plasma, 50 μL of HEV or SSV samples at 250 μg/mL (based on the concentration that produced maximal cytotoxicity in lymphoma cells), and 100 μL Tris–HCl buffer to a final volume of 200 μL. They were then incubated for 5 min at 37 °C, after which 10 μL of 150 mM CaCl2 were added for coagulative process re-activation, following the reaction during 23 min at 37 °C, and ODs were read in a microplate reader (Bio-Tek Instruments, Inc.) at 565 nm.
Statistical analysis
Results were expressed as means ± SD of triplicate determinations from three independent experiments. Statistical significance (p ≤ 0.05) was assessed by one-way analysis of variance and by the Student’s t test. More
