Antitumor activity of Chlorella sorokiniana and Scenedesmus sp. microalgae native of Nuevo León State, México

Reagents, culture media, and tumor cells

L-glutamine and penicillin-streptomycin solutions were purchased from Life Technologies (Grand Island, NY). Concanavalin A (Con A), RPMI 1640 medium, fetal bovine serum (FBS), sodium dodecyl sulfate (SDS), N, N-dimethylformamide (DMF), phosphate buffered saline (PBS), and 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) were obtained from Sigma-Aldrich (St. Louis, MO, USA). Vincristine was obtained from Vintec (Columbia, S.A. de C.V., Ciudad de México). Extraction buffer was prepared by dissolving 20% (wt/vol) SDS at 37 °C in a solution of 50% each DMF and demineralized water, and the pH was adjusted to 4.7. The tumor cell line L5178Y-R (mouse DBA/2 lymphoma) was purchased from the American Type Culture Collection (LY-R, ATCC^® CRL-1722™; ATCC, Rockville, MD, USA), 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 medium) at 37 °C, in a humidified atmosphere of 5% CO₂ in air. Cellular density was kept between 10⁵ and 10⁶ cells/mL.

Microalgae strains and culture

C. sorokiniana was isolated from San Juan River in the municipality of Cadereyta (26°21′55″N 98°51′15″O), whereas Scenedesmus was obtained from Pesquería River in the municipality of Apodaca (25°47′06″N 100°03′04″O) Nuevo Leon, Mexico. C. sorokiniana molecular identification using the P2F (5′-GGC TCA TTA AAT CAG TTA TAG-3′) and P2R (5′-CCT TGT TAC GA(C/T) TTC TCC TTC-3′) primers (Lee & Hur, 2009), which amplifies for a 1,700 bp fragment of the 18S gene, as previously reported by Cantú-Bernal (2017). Amplification conditions were an initial denaturation cycle at 95 °C for 5 min, 30–35 denaturation cycles at 95 °C for 30 s, alignment at 50–55°C for 30 s, and an extension process at 72 °C for 105 s, followed by a final extension at 72 °C for 7 min. The PCR product was confirmed by electrophoresis on 1.5% agarose gel at 100 Volts for 35 min, where the expected 1,700 bp band was observed. Once the PCR product was confirmed, the band was purified, for which the Wizard SV Gel and PCR clean-up system kit (Promega, Invitrogen) was used. For the band sequencing, the product was sent to the synthesis and sequencing unit of the Institute of Biotechnology, Universidad Nacional Autónoma de México. The edition and analysis of the Chlorella sp. sequence similarity percentage was carried out using the program Bioedit Sequence Alignment Editor v. 7.1.9 by sequence identity matrix means, after being compared with sequences reported in GenBank.

For microalgae culture, water samples were taken on 50 mL sterile Falcon tubes and kept at 5 °C ± 2 °C on ice. Then, 5 mL were transferred to 250 mL Erlenmeyer flasks, containing 100 mL of LC culture medium, as developed and reported by López-Chuken, Young & Guzman-Mar (2010). Flasks were then incubated at room temperature (25 °C ± 3 °C) in a continuous shaker at 120× g and under light radiation using 100 Watt white fluorescent light bulb as a continuous artificial light source (1,000 lux approximately). Flasks were incubated for 14 d until green growth was observed, after which, 100 µL were transferred to Petri dishes containing the same culture medium, but solidified with 1.5% of bacteriological agar. Inoculated dishes were incubated at 30 °C ± 2 °C by using a 100 watt white fluorescent light bulb as a continuous artificial light until isolated green colonies were observed. Single colonies were collected using a bacteriological loop and placed in Erlenmeyer flasks containing 100 mL of algal LC liquid culture medium. Next, flasks were incubated under the same conditions described above. This process allowed us selecting a single microalgae genus by picking up a single colony; however, given that microalgae tend to grow in consortia with bacteria and yeasts, microalgal cultures were treated with an antibiotic and antimycotic solution containing 500 UI/mL penicillin, 500 µg/mL streptomycin, 50 µg/mL gentamicin, and 1.25 µg/mL fungizone. For this, 5 mL of LC liquid culture medium with antibiotics were placed in 15 mL conical tubes, after which 0.25 mL of the algal culture were added and tubes were incubated for 48 h, under same shaking and lighting conditions described above. After the incubation period, 500 µL of the cultures were transferred into 50 mL of sterilized LC liquid culture medium without antibiotics producing axenic cultures of C. sorokiniana and Scenedesmus sp. isolates. Each axenic culture was grown for 14 d in 1-L Erlenmeyer flasks containing 500 mL of LC liquid culture medium, until exponential growth phase was reached (based on growth curve, Fig. S1). Next, each complete culture was transferred to individual bioreactor tanks containing 14.5 L of LC culture medium. Photobioreactor tanks were designed by the López-Chuken work team, and consisted of circular acrylic tanks of 30 cm of diameter and height; aeration was supplemented by air pumps with an adapted 0.2 µm filter at 1-L/min flow rate, radiated by continuous artificial LED white lights at 1,500 lux of intensity, and agitation by rotary plastic pallets at 50 rpm (Fig. S2). Biomass production in bioreactors was monitored every 2 d (Tuesdays, Thursdays, and Saturdays) by taking a 10 mL sample with a sterile pipette and filtering through a previously weighed 0.7 µm-pore size microfiber paper. Then the paper was dried at 70 °C inside an oven and weighed again; this monitoring process was repeated until the biomass production showed no increase. Once the maximum biomass production was reached, bioreactor tanks were stored at 4 °C ± 2 °C, until most microalgae biomass precipitated, then, the supernatant was decanted (Fig. S3). The collected wet biomass was the centrifuged at 9,000 rpm for 10 min (ST16R model; Thermo Fisher Scientific, Waltham MA, USA) and frozen dried (Labconco, Kansas City, MO, USA).

Biomass dried samples of C. sorokiniana and Scenedesmus sp. were placed in separate Whatman cellulose extraction thimbles (33 × 80 mm, thickness 1.5 mm) (Sigma-Aldrich, St. Louis, MO, USA) and placed in a Soxhlet extraction apparatus (Reyna-Martínez et al., 2015), which is a continuous system consisting of a flat bottomed round flask, an extraction chamber with a siphon, and a condenser. This method was selected since this extraction is very practical and recommended by most of the methanol-soluble compounds for biological material recovering. A round flask filled with 600 mL of methanol was used and the extraction lasted 48 h for each microalgae. Methanol was selected based on preliminary results where methanol extracts showed the highest cytotoxic activity against L5178Y-R cell line; whereas chloroformic extracts did not show cytotoxic effects and hexane itself showed cytotoxicity against the tumor cell line tested. After the biological material compounds were extracted with methanol, the solutions were filtered using Whatman filter paper, and solvent was evaporated using a rotary evaporator, leaving approximately 10 to 15 mL of liquid material. Remaining solvent was further removed by a vacuum desiccator. Extracts were dissolved in RPMI medium at a concentration of 1 mg/mL and kept frozen until use. From this stock, serial 1:1 dilutions from 500 to 7.8 µg/mL were prepared.

The tumor cell line L5178Y-R (mouse DBA/2 lymphoma) was purchased from the American Type Culture Collection (LY-R, ATCC^® CRL-1722™; ATCC, Rockville, MD, USA), 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 medium) at 37 °C, in a humidified atmosphere of 5% CO₂ in air. Cellular density was kept between 10⁵ and 10⁶ cells/mL.

Tumor cytotoxicity and apoptosis assays

To determine the cytotoxic effect of C. sorokiniana and Scenedesmus sp. methanol extracts against L5178Y-R tumor cells, cell cultures were collected and washed three times in RPMI medium, then suspended and adjusted to 5 × 10⁴ cells/mL with complete RPMI medium. One hundred microliters of the cell suspensions were then added to flat-bottomed 96-well plates (Becton Dickinson, Cockeysville, MD, USA), containing 100 µL of complete RPMI, methanol microalgae extracts at various concentrations, vincristine (250 µg/mL) as positive control, and RPMI medium as negative control; all treatments were tested in triplicate. Microplates were incubated for 48 h at 37 °C with 5% CO₂, then 15 µL of MTT were added (0.5 µg/mL, final concentration), and cultures were incubated for 3 additional hours. After this, supernatant was removed and 80 µL of DMSO were added to all wells. Optical densities, resulting from dissolved formazan crystals, were then read in a microplate reader (DTX 880 Multimode detector; Becton Dickinson, Schwechat, Austria) at 570 nm (Gomez-Flores et al., 2009). The percentage of cytotoxicity was calculated as follows: ${\displaystyle \text{\%}\text{Cytotoxicity}=100-\left[\left(A_{570}{\text{in extract-treated cells/A}}_{570}\text{in untreated cells}\right)\times 100\right].}$ Apoptosis induction by C. sorokiniana and Scenedesmus sp. methanol extracts against L5178Y-R cell line was evaluated in vitro by acridine orange and ethidium bromide staining. For this, 1 × 10⁶ L5178Y-R tumor cells were placed in 24-well plates in the presence of 500 µg/mL methanol extracts, and incubated for 24 h. Then, 500 µL of RPMI, plus 1-µL of acridine orange and 100 µg/mL ethidium bromide (1:1 ratio) were added to the wells. Next, cultured cells were incubated for 5 min, washed with 1-mL PBS, and suspended in 100 µL of RPMI medium; after incubation period, 10 µL of cell suspension were placed between a slide and a coverslip for fluorescence microscope visualization (Inverted Tissue Culture Fluorescence Microscope Olympus IX-70, Representaciones y Distribuciones FAL, S.A. de C.V., Naucalpan, State of México, Mexico). Acridine orange stains viable cells and dead cells (green cells), whereas ethidium bromide only stains those cells that have lost the integrity of their membrane (orange cells). Therefore, viable cells appear in a uniform green tone, cells found in apoptosis appear in a spotty green or granular in the center due to the condensation of chromatin and fragmentation of the nucleus, whereas cells in necrosis appear in a uniform orange hue (Coligan et al., 1995).

In addition, apoptosis induction was evaluated by DNA degradation (Orozco-Flores et al., 2017). For this, cells were incubated for 48 h with C. sorokiniana and Scenedesmus sp. methanol extracts at 500 µg/mL, testing their respective negative (culture medium) and positive (20 µg/mL Actinomycin D) controls. After the incubation period, cells were collected and centrifuged at 2,000 rpm for 10 min, then washed with PBS and extracted using the AxyPrep Multisource Genomic DNA Miniprep Kit (Axygen, Tewksbury, MA, USA). In order to visualize the extracted DNA, the sample was separated by 2% agarose gel electrophoresis, using SB buffer for the electrophoretic shift at 70 V for 20 min and 110 V for 1 h. After this, gel was stained with 5 ng/mL ethidium bromide and photographs were documented under High Performance Ultraviolet Transilluminator (UVP, LLC, Upland, CA, USA) light. DNA like-ladder fragmentation indicates apoptotic activity, whereas DNA smear represents cell death by necrosis.

In early apoptosis stages caspase enzymes are activated. Caspase participate in the cleavage of protein substrates leading to cell disassembly. Cleavage of protein substrates leads to a fluorescent monoamide formation and finally to a rhodamine 110 conversion. For apoptotic pathway involving caspases, caspase can be monitored by measuring fluorescence intensity using microplate wells (Towhid et al., 2013). For this, L5178Y-R cells (5 × 10⁵ cells/well) were seeded in a 48 wells plate, and treated with Actinomycin D (800 ng/mL) as positive control, or Chlorella and Scenedesmus methanolic extracts at 500 µg/mL. Cultures were then incubated for 24 h at 37 °C, after which, activated caspases were detected with the CaspGLOW™ red active caspase staining kit following manufacturer’s instructions. Fluorescence intensity was measured at Ex/EM = 540/570 nm in a Varioskan Lux Multimode Reader (Thermo Fisher Scientific, Waltham, MA, USA).

Animals

Six- to eight-week old Balb/c female mice were purchased from Harlan Mexico S.A. de C.V. (D.F., Mexico). They were kept in a pathogen- and stress-free 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, USA), and given water and food ad libitum. Animals were euthanized by asphyxiation in a 100% CO₂ chamber. Experiments involving the use of animals were reviewed and approved by our institutional animal care and use committee before being initiated, and were performed in accordance with the Guiding Principles in the Use of Animals in Toxicology, adopted by the Society of Toxicology in March 1999.

Murine thymus lymphocyte viability assay

Thymus was immediately removed after mouse death. Single-cell suspensions were prepared by disrupting the organ in RPMI 1640 medium. Cell suspensions were washed three times in this medium, and suspended and adjusted at 1 × 10⁷ cells/mL in complete RPMI medium. Thymus lymphocyte viability was determined by a colorimetric technique using MTT (Gomez-Flores et al., 2009). Thymus suspensions (100 µL of 1 × 10⁷ cells/mL) were added to flat-bottomed 96-well plates (Becton Dickinson, Cockeysville, MD, USA) containing triplicate cultures (100 µL/well) of complete RPMI medium (unstimulated control), or 100 µL of C. sorokiniana and Scenedesmus sp. methanol extracts at various concentrations, for 48 h at 37 °C in 95% air-5% CO₂ atmosphere. After incubation for 44 h, MTT (0.5 mg/mL final concentration) was added, and cultures were additionally incubated for 4 h. Cell cultures were then incubated for 16 h with extraction buffer (100 µL/well), and optical densities, resulting from dissolved formazan crystals, were then read in a microplate reader (Becton Dickinson, Cockeysville, MD, USA) at 570 nm (Gomez-Flores et al., 2009).

All experiments were repeated at least three times with similar results. The results were expressed as means ± SEM of triplicate determinations from a representative experiment. Statistical significance was assessed by one-way analysis of variance and by the Student’s t test.