Journal of Cancer Prevention 2017; 22(1): 40-46
Published online March 30, 2017
© Korean Society of Cancer Prevention
Hassan Akef1, Nahla Kotb1, Dina Abo-Elmatty2, and Sayed Salem3
1National Organization for Research and Control of Biologicals (NORCB), Giza, Egypt, 2Biochemistry Department, Faculty of Pharmacy, Suez Canal University, Ismailia, Egypt, 3Animal Health Research Institute (AHRI), Giza, Egypt
Correspondence to :
Hassan Akef, National Organization for Research and Control of Biologicals (NORCB), P.O. Box 354, Postal code 12611, Dokki, Giza, Egypt, Tel: +20-2-37480478, Fax: +20-2-37628892, E-mail: email@example.com, ORCID: Hassan Akef, http://orcid.org/0000-0002-0211-6096
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
The present study evaluated the effects of
Keywords: Venoms, Apoptosis,
Scorpion venom is a natural product that is a complex mixture of molecules and plays an important role in defense and prey capture. Some scorpion venoms show cytotoxic effects against a wide range of cancer cell lines. Indian black scorpion (
Another class of natural product is snake venom toxin, which has been reported to possess cytotoxic effects against cancer cell lines. A heat stable protein toxin (drCT-I) from Indian
Apoptosis is regulated by various protein families, including the
The present study evaluated the effects of scorpion venom from
The human prostate cancer cell line (PC3) was provided by VACSERA (Giza Governorate, Egypt). The cells were maintained at 37°C in a humidified incubator with 5% CO2 and RPMI 1640 media with L-glutamine (Lonza, Verviers, Belgium), which was supplemented with 10% heat inactivated FBS (Biowest, Nuaillé, France).
Scorpion venom from
The effect of the venoms studied (scorpion venom
After treatment of the PC3 cells with the IC50 (median inhibitory concentration) values of the venoms for 24 hours at 37°C in a humidified incubator with 5% CO2, the cells were harvested. The total RNA was isolated from treated and untreated PC3 cells using an RNeasy mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The concentration and purity of the isolated RNA were detected by NanoDrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA).
cDNA synthesis was performed using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) in 20 μL reaction mixture containing 10 μL RNA sample (1 μg), 2 μL 10× RT Buffer, 0.8 μL 25× dNTP Mix (100 mM), 2 μL 10× RT random primers, 1 μL MultiScribe Reverse Transcriptase, 1 μL RNase inhibitor, and 3.2 μL nuclease-free water. Reverse transcription was performed using thermal cycler (Applied Biosystems) with the following temperatures and times: Step 1, 10 minutes at 25°C; Step 2, 120 minutes at 37°C; Step 3, 5 minutes at 85°C; then Step 4, hold at 4°C.
SYBR Green quantitative real-time-PCR (qPCR) was performed on cDNA extracted from treated and untreated PC3 cells. The expression of target genes was quantified with SYBR Green PCR master mix using StepOne real-time PCR system (Applied Biosystems). Each qPCR amplification reaction was performed in 20 μL reaction mixture containing 10 μL Power SYBR Green PCR master mix (2×), 2 μL cDNA sample (100 ng), 1 μL forward primer (10 μM), 1 μL reverse primer (10 μM), and 6 μL double-distilled water. The cycling conditions started with initial 10 minutes at 95°C, followed by 40 two-step cycles of 15 seconds at 95°C and 1 minute at 60°C. The amplification was checked by melting curve analysis. The relative gene expression was calculated using 2−ΔΔCT method,9 where ΔΔCT values of each sample were calculated from CT values; ΔΔCT = [CT target gene–CT
PC3 cells were treated with the IC50 values of the venoms for 24 hours in a humidified atmosphere of 5% CO2 at 37°C. After that treatment, the cells (floating and adherent cells) were harvested, washed twice with cold PBS (by centrifugation), and pelleted at 2,000 ×
The MDA level and antioxidant enzyme (catalase [CAT], superoxide dismutase [SOD], glutathione peroxidase [GPx], glutathione reductase [GR], and glutathione-S-transferase [GST]) activity levels were, according to the manufacturer’s instructions, assayed in treated and untreated PC3 cells using readymade kits provided by Biodiagnostic (Cairo, Egypt) and Milton Roy spectronic 21D UV-Visible spectrophotometer (USA). The total protein was determined in PC3 cells by the Bradford method.12 The MDA and the antioxidant enzyme activity levels were calculated per mg protein.
All experiments were independently performed at least three times. The IC50 values were determined using GraphPad Prism7 (GraphPad software, La Jolla, CA, USA). The
The present work evaluated the changes that occurred in a human PC3 cells treated with the venoms studied. Many reports have demonstrated the cytotoxic, anti-proliferative, and apoptotic effects of different snake and scorpion venoms against different cancer cell lines.1,4 This evidence, like our results, supports the potential of these venoms to act against an epithelial cancer cell line. The venom’s effect on the viability of PC3 cells was evaluated by an MTT assay. We observed that treatment with these venoms resulted in a dose-dependent decrease in the PC3 cell viability (Fig. 1). The IC50 values for
Androgen-independent prostate cancer cells, such as PC3 cells, show an increase in
The changes that occurred in oxidative stress were tested in PC3 cells after venom treatment. Generally, oxidative stress is due to the imbalance between the antioxidants and pro-oxidants in favor of the oxidants.25 Venom/toxin-induced oxidative stress results in increased levels of oxidative markers,6,7 and it sometimes causes malfunctioning of vital organs through membrane destruction, enzyme release, and protein loss.26 Lipid peroxidation was measured by the MDA level, which is a biomarker of oxidative stress and cellular damage, that is evoked by stressors. A 24 hours treatment of PC3 cells with the IC50 of the venoms caused a significant increase in MDA in the cell lysate compared to the control cells (Table 1). This agrees with reports that showed an elevation of lipid peroxidation levels after treatment with various types of venoms,27,28 and increased lipoperoxidation may result in membrane damage. In the defense against oxidative stress, the cellular antioxidant enzyme system plays an important role. This system includes CAT, SOD, GPx, GR, and GST (the antioxidant enzymes studied). CAT converts H2O2 to H2O, SOD catalyzes the dismutation of the superoxide radical anion, GPx catalyzes GSH (the reduced form of glutathione) oxidation to oxidized glutathione (GSSG) at the expense of hydrogen peroxide or other organic peroxides, GR recycles GSSG back to GSH using NADPH, and GST catalyzes the conjugation of GSH to xenobiotic substrates for detoxification of nucleic acids. The induction of antioxidant enzymes is necessary for the host defense to protect the cell against oxidative stress. Antioxidant enzymes are working simultaneously to prevent the formation of highly cytotoxic hydroxyl radicals. The antioxidant defense system in PC3 cells was tested in response to venom. Our results show that a 24 hours treatment of PC3 cells with the IC50 of these venoms caused a significant increase in the activity of the antioxidant enzymes in the cell lysate compared to the control cells (Table 1). This agrees with reports that showed an increase in lipid peroxidation level and antioxidant enzymes when using venom as a treatment.28,29 In addition, da Silva et al.7 reported increased activities of CAT and GST in venom-injected experimental animals, which also agrees with our results. However, in contrast to our results, other studies showed decreases in some antioxidant enzymes in case of treatment with venoms.30
In conclusion, the venoms studied have cytotoxic and anti-proliferative activities against PC3 cells.
The authors gratefully thank Dr. Aly Fahmy Mohamed (VACSERA, Egypt) for his valuable technical advises.
Effect of the venoms at the IC50 value on the activity of the antioxidant enzymes and MDA level in PC3 cells.
|Variable/mg protein||Control cell||PC3 cells treated with|
|Mix of |
|CAT (mU)||0.84 ± 0.10||1.49 ± 0.12 (0.02)||2.90 ± 0.33 (0.04)||4.00 ± 0.27 (0.003)|
|SOD (U)||168.75 ± 0.81||232.20 ± 9.01 (0.03)||385.75 ± 32.78 (0.03)||638.24 ± 41.81 (0.01)|
|GPx (mU)||4.60 ± 0.31||7.64 ± 0.48 (0.02)||14.05 ± 0.49 (< 0.001)||13.03 ± 0.38 (0.0002)|
|GR (mU)||17.82 ± 0.43||27.73 ± 0.79 (0.003)||47.29 ± 4.01 (0.03)||86.27 ± 7.34 (0.02)|
|GST (mU)||3.09 ± 0.10||5.22 ± 0.26 (< 0.01)||8.97 ± 0.29 (0.004)||9.68 ± 0.24 (0.0002)|
|MDA level (nmol)||4.43 ± 0.20||6.77 ± 0.49 (0.04)||14.17 ± 0.90 (0.01)||16.17 ± 1.13 (0.01)|
Values are present as mean ± SE (
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