POTENTIAL ANTI CANCER ACTIVITY OF SNAKE VENOM, BEE VENOM AND THEIR COMPONENTS IN LIVER AND BREAST CARCINOMA

POTENTIAL ANTI CANCER ACTIVITY OF SNAKE VENOM, BEE VENOM AND THEIR COMPONENTS IN LIVER AND BREAST CARCINOMA

Fathia Zaky El Sharkawi ^*1, Shaimaa Saber Saleh ² and Aly Fahmy Mohamed El Sayed ²

Department of Biochemistry and Molecular Biology ¹, Faculty of Pharmacy, Helwan University, Cairo, Egypt

The Egyptian Holding Company for Biological Products and Vaccin (VACSERA) ^{2, 3}, Cairo, Egypt

ABSTRACT: The present study aimed to evaluate the antitumor efficacy of Snake venom (SV), bee venom (BV), combinations of their components and their apoptotic, cell death mechanisms in liver (HepG2) and breast (MCF7) cell lines. Cytotoxic effects of venoms, L-Amino acid oxidase (svLAAO), phospholipase A2(svPLA2), Melittin ( MEL) were tested against HepG2 and MCF-7 cell lines and IC50 was calculated . mRNA expression of p53, Bax and Bcl-2  was carried out. All the tested compounds had anti proliferative effects on the tumor cell lines with different potency. BV had a higher cytotoxicity against liver and breast cells (IC50=1.26 and 2.85 µg/ml) than SV (IC50=5.86, 13.05µg /ml). The cytotoxicity of svLAAO was much higher than SV with IC50 = 3.65 and 0.48 µg /ml. MEL showed a higher cytotoxic effect than BV against MCF7 cells (IC50=1.14 µg /ml). MEL when combined with svPLA2 gave a synergistic effect on the expression of P53 and Bax in the two cancer cells. Venoms combination had the least synergistic effect relative to other combinations. From our results it could be concluded that SV, BV and their major components are promising anticancer agents for liver and breast carcinoma. SvLAAO is the most effective and safe compound (than its crude venom) to be used as antitumor agent or in combination with chemotherapeutic drug. MEL is a good candidate for liver and breast cancer treatment, it could be used in combined form with svPLA2 or svLAAO

Venoms,

Combinations,

cytotoxicity,

HepG2, MCF7,

apoptotic genes

INTRODUCTION: Cancer is the major public burden in all developed and developing countries. A total of 1,638,910 new cancer cases and 577,190 deaths from cancer are projected to occur in year 2012 ¹. In all types of cancer, genetic alterations give rise to changes in expression, activation or localization of regulatory proteins in the cells, affecting the signaling pathways that alter their response to regulatory stimuli and allow the unrestricted cell growth.

Various therapies have been used for treating cancer such as chemotherapy, radiotherapy, immunotherapy and gene therapy ²; but still there is an urgent need of finding a better natural safe way to treat cancer with little effect on normal cells. Anticancer drug developments from natural biological resources are ventured throughout the world.

The biodiversity of venoms or toxins made it a unique tool from which new therapeutic agents may be developed. Several compounds from venomous animals, such as snakes, spiders, scorpions, caterpillars, bees, insects, wasps, centipedes, ants, toads, and frogs, have largely shown biotechnological or pharmacological applications ^{3, 4, 5, 6, 7}. This leads to search for cancer cure from natural products.

Venom is a secretion of venomous animals synthesized in venom gland. It is a modified saliva containing a mixture of different bioactive proteins and polypeptides used by an animal for defense or to immobilize its prey ⁸. Snake venoms consist of a mixture of proteins with or without catalytic activity such as phospholipase A2 (PLA2), proteases, hyaluronidases, L-amino acid oxidases (LAAOs), acetylcholinesterases, growth factors, protein C activators, lectins, and von Wille brand factor-binding proteins; peptides mainly comprising bradykinin potentiators and disintegrins; also it contains low molecular weight organic compounds (carbohydrates, serotonin, histamine, citrate, and nucleosides) and inorganic ions such as (calcium, cobalt, magnesium, copper, iron, and potassium) as well as enzymatic inhibitors ⁹.

Not only the venom of every snake is different but a subtle difference exists between different species, between juveniles and adults, even among the snake of same species but of different geographical regions. Approximately 90-95% of venom’s dry weight is composed of proteins that may be toxic or non-toxic ¹⁰.The ability of snake venoms to act upon tumor cells has been known for a long time. The first reported studies on using snake venom against tumor cells were related to the defibrination process ¹¹.

L-amino acid oxidase (LAAO) constitutes 1-9% of the total venom protein and is responsible for the light yellowish color of the venom ¹² it has been reported to exhibit a wide range of pharmacological activities including anti-proliferative ¹³ and anti-bacterial activities ¹⁴. It can induce apoptosis in mammalian endothelial cells due to the production of high concentration of hydrogen peroxide which kills the cells ¹².Phospholipase A₂  (PLA2) plays an important role in many biological events such as cell signaling and cell growth, generation of pro-inflammatory lipid mediators such as prostaglandin, and leukotrienes ¹⁵.

Snake venoms are the richest sources of PLA2 which display a variety of relevant toxic actions such as neurotoxicity, cytotoxicity, cardiotoxicity, hypotensive and proinflammatory effects ^{16, 17}. Bee venom (BV) contains a variety of different peptides, including melittin, phospholipase A2, apamin, adolapin and mast cell-degranulating peptide (MCDP); it also contains non-peptide components including lipids, carbohydrates and free amino acids, all having many cellular activities ¹⁸. BV has been used as a traditional medicine to treat back pain, rheumatism, and skin diseases due to its antibacterial, antiviral, and anti-inflammatory effects ¹⁹. Several studies have demonstrated that bee venom either or melittin have anti-proliferative effects on various cancer cells such as prostate,liver, breast, cervical and renal cancer cells through intrinsic or extrinsic apoptosis ^{20, 21, 22, 23}.

Melittin (MEL) is a major peptide constituent of bee venom that has been proposed as one of the upcoming possibilities for anticancer therapy. Recent reports point to several mechanisms of MEL cytotoxicity in different types of cancer cells such as cell cycle alterations, effect on proliferation and/or growth inhibition, and induction of apoptotic and necrotic cell death trough several cancer cell death mechanisms, including the activation of caspases and matrix metalloproteinases ²⁴.

However, little is known about the anticancer activity of  crude SV,BV and their venomous major components    against liver and breast cancers which are the most spread types worldwide. Also there is no information about the synergistic or antagonistic  anticancer effects of the combined treatment with venoms and venomous materials .So the current study aims to evaluate the anticancer activities of  crude BV,SV , their major components ( LAAO,PLA2 ,MEL)  as well as their combinations against liver and breast cancer cells and also  the effects of  these tested compounds on the  expression of the apoptotic genes and on the cell cycle of the tested tumor cells.

MATERIALS AND METHODS:

Materials:

Chemicals:

RPMI 1640 medium with L- glutamine (Biowhittaker - Belgium), Fetal bovine serum (FBS) (GIBCO - USA), dimethyl sulfoxide (DMSO) solvent and other cell culture materials were purchased from Fisher Scientific Cell Culture (Houston, TX, USA). Other reagents were of the highest analytical grade available.

Culture cells and venoms:

Human breast adenocarcinoma cells (MCF7 - ATCC number: HTB-22), human hepatocellular carcinoma cells (HepG2 ATCC number HB-8065), non tumorigenic normal human lung fibroblast (MRC-5 cells – ATCC No. CCl-171), snake venom: Naja haje (Egyptian copra) and bee venom from honey bee Apis mellifera were supplied from Vaccera sera plant (Egypt).

L-Amino acid oxidase  from snake venom  (svLAAO) , svPLA2 from snake venom 1,500 units/ mg protein (lyophilized powder ) and  Melittin ( MEL) from honey bee venom: Apis mellifera, Purity 96.50%  were purchased from Sigma company(Sigma-Sain Diago- USA ).

RNA and apoptotic genes:

Bioopure RNA isolation reagent were purchased from Bio Scientific Group Austin, USA. Maxima first strand cDNA synthesis Kit for RT-q PCR and Primer sequences of P53, Bax and Bcl-2 genes were supplied from Thermo Scientific USA.

Cytotoxicity assays:

Cytotoxic effects of venoms( Snake and Bee ), L-Amino acid oxidase(LAAO), PLA2 and MEL were tested against MCF- 7, HepG2 and MRC-5   in vitro cell lines by the MTT assay as previously described by Alley et al., 1988 ²⁵. Negative cell control was included. IC50 was calculated by using Masterplex 2010 hitachia (GIRSS). Data were presented as IC50 of the tested compounds compared to that of normal cell control MRC-5 Five combinations of venoms and venomus components were prepared from the resulted IC50 value of each   compound for evaluation of their apoptotic effects on the tested tumor cell lines. These combinations are: BV + SV (combination 1),

LAAO+PLA2(combination2),LAAO+MEL(combination3),PLA2+MEL(combinations4),MEL+LAAO+PLA2(combination5).

Apoptotic evaluation:

RNA was extracted from 24 hr treated cells with venoms, LAAO   PLA2, MEL, the 5 combinations mentioned above, treated and untreated control cells by using RNA isolation kit according to manufacturer's protocol. Extracted RNA was reverse transcripted to cDNA using Maxima first strand cDNA synthesis Kit. The mRNA expression of pro-apoptotic genes (P53 and Bax) and anti-apoptotic gene (Bcl-2) was carried out using the newly synthesized cDNA as template for PCR. Semi-quantitative RT-PCR was carried out according to ²⁶.  Using the specific primer for each gene (in a concentration of 10pmole/ul for each primer) and housekeeping gene (GAPDH).

Gel electrophoresis for PCR products (10µl) was carried out on 1.5% agarose gel using DNA ladder 1.5 KB and visualized using UV transillumiator after staining with ethidium bromide followed by denstiometric analysis of bands intensities which expressed as relative absorbance units. Data representing mRNA expression levels of p53, Bax and Bcl-2 were calculated as band intensities compared to GAPDH by using alpha gel documentation system.

Cell cycle analysis:

Cell cycle analysis is carried out according to the method of (Mansouri  M et al 2014 )Each cell line type  (MCF7 , HepG2 and MRC5  ) was seeded into 96-well plates at a density of 6 x10⁵ cells/well for 24 h. Then, the cells were treated with 100 mM  SV, BV ,  LAAO and MEL for 24 h. Approximately 2.5 x 10⁵ cells were harvested, washed twice using ice-cold PBS, and re suspended in 200 ml of cold PBS (phosphate –buffered saline). For rapid cell fixation and dispersion, the cell suspensions were added drop wise to 1 ml of cold 70% ethanol and incubated on ice for 45 min.

After centrifugation, the cells were resuspended in 1ml of propidium iodide (PI) master mix (PBS containing 100 mg/ml RNase A and 40 mg/ml PI) and incubated in the dark at 37 ºC for 30 min. The various cell cycle phases were monitored using a FACSCalibur_ flow -cytometer (BD Biosciences, San Jose, CA). Cells were excited at 488 nm with an argon laser, and the emission from ten thousand cells was recorded using a 580 nm band-pass filter (FL2-H) ²⁷. The obtained cell cycle profiles were analyzed using CellQuest version 3.2 and Win MDI version 2.8 softwares.

RESULTS:

1-**Cytotoxic effects of crude Snake and Bee venoms against MCF7 and HepG2 cells: The tested concentration is 100ug/ml

Both venoms have an anti proliferative effects on HepG2 and MCF7 cell lines. Bee venom shows  a significant high cytotoxic  activity against both types of tumor cells  exceeds that to normal cell control MRC-5 with 50% inhibition concentrations (IC50) of  1.26 and 2.85µg /ml respectively compared to MRC-5 (77.7µg /ml ) while snake venom exerts a different effect; it affect  the proliferation of the  liver cancer cells (IC50=5.86 µg /ml)  more than the breast cells IC50 = 13.05µg /ml compared to  MRC-5 cells (IC50=26.118µg /ml), (Table 1, Fig. 1, 2).

1-A** Cytotoxic effects of LAAO, PLA2:

LAAO as one of the components of snake venom  shows more potent inhibitory activity than the crude  snake venom on both HepG2 cells (IC50=3.65 µg /ml) and MCF7( IC50 =0.487 µg /ml) compared to MRC-5 normal cells (IC50=140.7µg /ml) in the tested concentration of 100 µg /ml while  the  opposite is shown with PLA2 since it gives a much lower cytotoxic effect than the crude venom on liver ( IC50 =53.6µg /ml)  and breast (27.6 µg /ml ) cancer cells compared  to normal cells ( IC50= 8.46µg /ml) in the tested concentration of 20ug/ml ( Table 1, Fig.2-4).

1-B** Cytotoxic effect of Melittin: (tested concentration is 20µg/ml)

Melittin as the main component of Bee venom shows high activity on MCF7 ( IC50 =1.14µg /ml) than the crude  bee venom in contrast to its effect on  HepG2 cells (IC50=6.12µg /ml) , on the other hand  the IC50 of  MEL  on MRC-5  cells (IC50=8.1µg /ml ) is higher that of the crude venom (Table 1 and Fig. 3, 4 ) .

TABLE 1: IC50 VALUES OF VENOMS AND VENOMOUS COMPONENTS IN MCF7, HEPG2 AND MRC5 TREATED CELL LINES.

FIG.1: EFFECT OF   SV, BV, LAAO ON CELL VIABILITY OF MCF-7 AND MRC5 CELL LINES. RESULTS ARE EXPRESSED AS PERCENTAGE OF VIABLE CELLS. CELLS WERE SEEDED IN 96 –WELL TISSUE CULTURE PLATES AND TREATED AFTER 24H WITH BV, SV, AND LAAO. CELLS WERE INCUBATED FOR 72H, PERCENT OF CELL VIABILITY WAS CALCULATED IN RELATION TO THE UNTREATED CONTROL. MTT COLORIMETRIC ASSAY METHOD WAS USED FOR DETERMINATION OF CELL VIABILITY.

Apoptotic effects of crude venoms and their components:

On the molecular level the effects of the tested compounds on mRNA expression of proappoptotic (p53, Bax) and anti appoptotic (Bcl2) genes in MCF7 and HepG2 cancer cells were determined semiquantitatively by RT-PCR followed by densitometrical analysis of the resulted gel electrophoresis bands.

All the tested compounds increase the expression of p53 and decrease that of Bcl2 in the treated cells with different potency (Fig. 5, 7, Table 2).  Concerning the expression of Bax, variable results have been observed. MEL does not increase the expression of Bax ( negative effect) in the two treated cells, while  SV has a positive effect (increase in  expression)  on Bax expression in MCF7 and a negative effect  in HepG2 cells .The remain tested compounds have positive activity on the two treated  cancer cells (Fig.6, Table 2).

FIG. 2:   EFFECT OF   SV, BV, LAAO ON CELL VIABILITY OF HEPG2AND MRC5 CELL LINES.

FIG.3: EFFECT OF   MEL AND PLA2 ON CELL VIABILITY OF MCF7 AND MRC5 CELL LINES. RESULTS ARE EXPRESSED AS PERCENTAGE OF VIABLE CELLS. CELLS WERE SEEDED IN 96 –WELL TISSUE CULTURE PLATES AND TREATED AFTER 24H WITH BV, SV, AND LAAO.CELLS WERE INCUBATED FOR 72H, PERCENT OF CELL VIABILITY WAS CALCULATED IN RELATION TO THE UNTREATED CONTROL. MTT COLORIMETRIC ASSAY METHOD WAS USED FOR DETERMINATION OF CELL VIABILITY.

FIG.4: EFFECT OF   MEL AND PLA2 ON CELL VIABILITY OF HEPG2 AND MRC5 CELL LINES.

FIG.5: EFFECT OF VENOMS AND VENOMOUS COMPONENTS ON THE EXPRESSION OF P53 GENE IN LIVER (A) AND BREAST (B) CANCER CELL LINES. RNA WAS EXTRACTED FROM 24 HR TREATED CELLS, TREATED AND UNTREATED CONTROL CELLS .THE MRNA EXPRESSION OF P53 GENE WAS CARRIED BY SEMI-QUANTITATIVE RT-PCR METHOD FOLLOWED BY GEL ELECTROPHORESIS FOR PCR PRODUCTS AND DENSITOMETRIC ANALYSIS OF BANDS INTENSITIES WHICH EXPRESSED AS RELATIVE ABSORBANCE UNITS. DATA REPRESENTING MRNA EXPRESSION LEVELS OF P53 WAS CALCULATED AS BAND INTENSITIES COMPARED TO GAPDH BY USING ALPHA GEL DOCUMENTATION SYSTEM.

FIG. 6: EFFECT OF VENOMS AND VENOMOUS COMPONENTS ON THE UP-EXPRESSION OF BAX GENE IN LIVER (A) AND BREAST (B) CANCER CELL LINES. DATA REPRESENTING MRNA EXPRESSION LEVELS OF BAX WAS CALCULATED AS BAND INTENSITIES COMPARED TO GAPDH BY USING ALPHA GEL DOCUMENTATION SYSTEM.

FIG.7: EFFECT OF VENOMS AND VENOMOUS COMPONENTS ON THE DOWN-EXPRESSION OF BCL-2 GENE IN LIVER (A) AND BREAST (B) CANCER CELLS. DATA REPRESENTING MRNA EXPRESSION LEVELS OF BCL-2 WAS CALCULATED AS BAND INTENSITIES COMPARED TO GAPDH BY USING ALPHA GEL DOCUMENTATION SYSTEM.

TABLE 2: MRNA EXPRESSION LEVELS OF APOPTOTIC GENES POST 24H TREATMENT OF LIVER AND BREAST CANCER CELLS WITH SV, BV, LAAO, PLA2, MEL AND THEIR COMBINATIONS. L+P=COMBINATION OF LAAO WITH PLA2, L+M= LAAO+MEL, P+M= PLA2+MEL, M+L+P= MEL +LAAO+PLA2

Synergistic and/or antagonistic apoptotic activity of combined treatment with venoms and venomous components:

Five combinations were tested for assessment of their synergistic and or antagonistic regulatory effects on the expression of apoptotic genes. Combination 2 (LAAO+PLA2) shows equal synergistic activity on the expression of pro and anti apoptotic genes in liver and breast cancer cells; while addition of MEL to LAAO or PLA2 (combinations 3and 4) gives different effects.

MEL causes an increase of the activity of PLA2 (combination  4)  and gives the highest synergistic  activity on the expression of p53, Bax genes in breast and liver cancer cells as well as BCl2   in liver cancer cells, while LAAO efficiency does not affected by combining with MEL .

Combination 5 (MEL + LAAO + PLA2) shows high synergistic effect on the down expression of Bcl2 in liver cancer cells with contrast effect on breast cancer cells (lowest synergistic effect).

The crude venoms combination shows variable effects in liver cells, it gives the least synergistic effect on p53 and Bcl2 than the effect of individual venom either SV or BV, but the contrast is shown with the up regulation of Bax since addition of BV increases the apoptotic effect of SV and leads to increase of Bax expression.  Table 2 and Fig. 8, 9, 10, 12 show the different potential apoptotic effects of the tested combinations in the treated cancer cells.

FIG. 8: EFFECT OF VENOMS AND VENOMOUS COMPONENTS COMBINATIONS ON THE EXPRESSION OF p53 GENE IN (A) LIVER AND (B) BREAST CANCER CELLS. CONT.: control (untreated cells), BV+SV=Bee Venom and Snake Venom combination L+p=svLAAO+svPLA2 combination, P+M=svPLA2+MEL combination, L+M=svLAAO +MEL combination, M+L+P= MELL +svLAAO + svPLA2 combination. Data representing mRNA expression levels of P53 was calculated as band intensities compared to GAPDH by using alpha gel documentation system.

FIG.9: EFFECT OF   VENOMS COMBINATIONS ON THE UP-EXPRESSION OF BAX GENE LIVER IN (A) AND BREAST (B) CANCER CELLS .

FIG. 10: EFFECT OF VENOMS COMBINATIONS ON THE EXPRESSION OF BCL-2 GENE IN LIVER (A) AND BREAST (B) CANCER CELL LINES.

FIG. 11: MRNA EXPRESSION OF APOPTOSIS RELATED GENES IN HEPG2 AND MCF7 CELLS POST TREATMENT WITH SV, BV, LAAO, PLA2 AND MELITTINE (M)

HepG2: lanes 1-5, MCF7 (lanes 6-10), Lanes 11, 12:  untreated control cells of HepG2 and MCF7 cells. Lad: DNA marker

FIG. 12: MRNA EXPRESSION OF APOPTOSIS RELATED GENES IN HEPG2AND MCF7) CELLS POST TREATMENT WITH DIFFERENT VENOMS AND VENOMOUS COMPONENTS COMBINATIONS.

Cell cycle arrested at theG0/G1 phase by tested venoms

All the tested venoms and venomous components arrest the treated cells at G0/G1 phase.  Fig.13 demonstrate the different percentage of cell cycle arrest at G0/G1 phase of 24h treated cells with venoms and venomous components.

FIG.13: FLOWCYTOMETRIC ANALYSIS POST 24 H TREATMENT OF HEPG2 AND MCF7 WITH VENOMS, VENOMOUS COMPONENTS AND THEIR COMBINATIONS

DISCUSSION: Although the use of chemotherapeutics in cancer therapy remains the predominant option for clinical control; it provides inadequate effect in addition to its effect on the normal cells as well as on cancer cells; also one of the major problems of chemotherapy is the resistance developed after initial treatment.

This has led to the increased demand of using anticancer drugs developed from natural resources. The biodiversity of venoms and toxins makes them a unique source from which novel therapeutics may be developed. Snake and bee venoms reported to have a potential cytotoxic effects on tumor cells [28]. However, little is known about their cytotoxicity as crude venoms compared with their major components (LAAO, PLA2, MEL) in breast and liver carcinoma which are considered to be the most spread cancer types worldwide. In the current study the anti proliferative effects of SV (copra), BV, svLAAO svPLA2 and MEL have been shown on liver and breast cancer cells. Snake venom contains cystatin (a member of cysteine protease family inhibitors) which has been reported to play an important role in tumor invasion and metastasis. In a study carried out on MHCC97H (liver cancer) cells, sv-cystatin has shown inhibition of tumor cell invasion and metastasis through the reduction of the proteinases activity and epithelial-mesenchymal transition (EMT) ²⁹.

On the other hand the cytotoxins from  some snake species(N. haje ,N. oxiana, and N. kaouthia)  can readily penetrate into living cancer cells as  human lung adenocarcinoma A549 ,promyelocytic leukemia HL60 and markedly accumulate in lysosomes (which are the main targets of cytotoxins) and cause lysosomal leakage and plasma membrane injury ³⁰. However there are many toxins from snake venom formulated into drugs for the treatment of cancer, hypertension and thrombosis ^{31, 32, 33, 34}; the potency of venom and its effect on human depend on the type and amount of venom injected and the site where it is deposited ³⁵.

The current  study shows the  potent efficacy of svLAAO  on breast and liver tumor cells (more  that of the crude SV) which is in agreement with different studies that isolate  LAAO and reported its cytotoxicity in stomach, colorectal, adenocarcinoma , human fibroblast cell lines ³⁶ , MCF7 and CACO-2 cancer  cell lines ³⁷, and on breast and lung tumorigenic cells ³⁸.

The present study supports a large number of studies that demonstrated the antitumor properties of BV and in particular of its major constituent MEL. Several mechanisms of BV cytotoxicity in different types of cancer cells have been reported such as cell cycle alterations, effect on proliferation and/or growth inhibition, and induction of apoptosis or necrosis ^{39, 40, 41, 42, 43, 44, 45}. Also, the anti proliferative effects of BV has been recently demonstrated on the cervix C33A cancer cells ⁴⁶ and lung cancer cells ^{47, 48}. On the other hand the anticancer activity of MEL has been approved since 2008 ⁴⁹ on HCC cells in nude mouse modles .MEL shares its amphipathic properties as one of the peptides isolated from BV that are characterized by their capacity to disturb tumor cell membrane bilayer integrity, either by creation of defects, disruption, or through pore formation with opening in the lipid bilayer leads to the collapse of transmembrane electrochemical gradients. In contrast to normal cells with low membrane potential, tumor cell membrane has a large membrane potential ^{50, 42, 51} therefore  many lytic peptides selectively disrupt the membrane structure of tumor cells to a greater extent than the membrane of normal cells ⁵¹.

Although MEL shows a potent cytotoxicity against tumor cells in the present study, it has   also a remarked toxicity to normal cells exceeds that of all tested components; this result is in agreement with (Soman et al., 2009) and (Pan et al., 2011) in that MEL is also toxic to normal cells and its therapeutic potential cannot be achieved without a proper delivery vehicle ^{52, 53}. On the other hand it was approved that MEL does not prevent the growth of normal cells at a concentration which prevents the proliferation of tumor cells ⁵⁴ therefore  it could play a useful role in the anticancer treatment ⁵¹.

The regulation of apoptosis in normal and malignant cells has become an area of extensive study in cancer research ⁵⁵ since the apoptotic process is involved in the growth and inhibition of the tumor cells. In the present study we examine the apoptotic regulatory effect of the tested venoms and their components as well as their combinations in liver and breast cancer cells through the increase in expression intensity of P53, Bax and decrease in that of Bcl2 resembling that approved by different studies on the relation between venoms and their cancer cell death by apoptotic process. The effect of BV on human lung cancer cells ^{47, 48} and cervix cancer C33A cells ⁴⁶ was reported through enhancement of death receptor (DR) expressions with increase in expression of pro-apoptotic proteins, up-regulated Bax and decrease in Bcl-2.

NF-κB. On the other hand similar studies reported the inhibitory effect of SV toxin on the growth of colorectal cancer cells through induction of intrinsic or extrinsic apoptosis ⁵⁶ and DR mediated apoptosis ⁵⁷. Also the apoptotic cell death of MEL has been reported against different in vitro tumor cell lines as in human hepatoma and glioma cell lines ^{41, 58}, hepatocellular carcinoma and osteosarcoma cell lines ^{59, 60, 61}. Furthermore MEL prevents liver cancer metastasis through inhibition of the Rac1- dependent pathway ⁴⁹; also it induces apoptosis in leukemic cells through the up-regulation of Bax and caspase-3 activation, down-regulation of Bcl-2 and the inhibitor of apoptosis protein (IAP) family members ⁶².

The apoptotic effect of LAAO also reported in human prostate adenocarcinoma (PC-3) model through increase in caspase-3/7 cleavage ³⁸.

Few studies reported the possible anti tumor effects of either venom and venom derivative combinations or their combinations with chemotherapeutic drugs.  To our knowledge the current study is the first one which investigate the possibility of using the tested venoms and /or their components in combined form as anticancer agents for liver and breast cancer. Lipps BV 1999, reported the synergetic activity of SV proteins atroporin and kaotree against cancer cells ⁶³. On the other hand the most important benefits of using the combination of a chemotherapeutic drug with venom or venomous component is to reduce the required therapeutic dose of either one , minimize their  side effects and also decrease the cancer cell resistance ; this information was recently confirmed  by (Gajskia G et al.2013) in his study on the  synergistic effect of using BV in combination with cisplatine on human cervical and laryngeal carcinoma cells  which could be useful in minimizing the cisplatin concentration during chemotherapy, consequently reducing and/or postponing the development of cisplatin resistance ⁶⁴.

The current study shows the synergistic  apoptotic effect of using PLA2 in combination with MEL in liver and breast cancer cells  which may be explained  by the proposal of (Holle et al., 2003 and Moon et al., 2006) that BV and its peptide components are related to the activation of PLA2, caspase and matrix metalloproteinase that destroy tumor cells ^50,42; also (Son et al., 2007) reported  that  MEL increases the activity of PLA2 on rat oncogen transformed cells resulting in their selective destruction ⁵¹. However, MEL is considered to be a very attractive candidate for cancer chemotherapy because cancer cells are less likely to develop resistance to a membrane pore-former so the combination of a chemotherapeutic drug together with MEL could be useful synergistic therapy ⁶⁵. ON the other hand MEL could be used in hepatocelleular carcinoma since it can synergies with TNF-related apoptosis-inducing ligand (TRAIL) in the induction of hcc apoptosis by activating the TAK1-JNK/p38 pathways ⁶⁶.

Regarding the use of crude venoms combination, the present study shows that SV and BV combination gives the least pro-apoptotic regulatory effect on the expression of p53 and Bax genes in the two tested cancer cells which may be due to certain antagonism/ interaction between venoms components when used in combined form.

Conclusions and future suggestions:

Finally it can be concluded that: SV, BV and their major components are promising anticancer agents for liver and breast carcinoma. sv LAAO  is  the most effective  and safe compound ( than its crude venom) to  be used as antitumor agent or in combination with chemotherapeutic drug. On the other hand MEL is a good candidate for liver and breast cancer treatment, it could be used in combined form with svPLA2 or svLAAO.

It can be recommended that more in vitro  animal studies are required to identify the  suitable therapeutic  safety dose of  these venomous compounds or combinations and their  possible side effects on normal cells., also several trials are recommended to deliver these compounds in a suitable pharmaceutical form (as nanoparticles) which could  minimize  their toxicity ,target the tumor cells  and facilitate their separate use or in combination with other chemotherapeutic agents.

AKNOWLEDGMENTS: The authors  thank Dr. Mohamed Rabie  the manager of polio production planet in the Holding Company for Biological Products and Vaccines ( VACSERA)  Egypt for  his assistance  in practical experiments and statistical analysis.

COMPETING INTERESTS: The authors declare that they have no competing interests.

REFERENCES:

1. Siegel R, Naishadham D, Jemal A .Cancer statistics. A Cancer J for Clin 2012; 62: 10-29.
2. Baskar R, Lee KA, Yeo R, Yeoh KW. Cancer and radiation therapy: current advances and future directions. Int J Med Sci 2012; 9 (3):193-9.
3. Lewis R J and Garcia M L. Therapeutic potential of venom peptides.Nature Reviews Drug Discovery 2003; 2: 790–802.
4. Fernandes-Pedrosa M F, Félix-Silva J and Menezes Y A S. Toxins from venomous animals: gene cloning, protein expression and biotechnological applications. In An Integrated View of the Molecular Recognition and Toxinology: From Analytical Procedures to Biomedical Applications. Chapter 2. Edited by Gandhi Rádis Baptista: InTech; 2013:23-71
5. Warner R L, McClintock S D, Barron A G and de la Iglesia F de. Hemostatic properties of a venomic protein in rodent dermal injuries. Experimental and Molecular Pathology 2007; 83: 241–248.
6. Warner R. L, McClintock S. D, Barron A. G, and de la Iglesia F. A, “Hemostatic properties of a venomic protein in rat organ trauma.Experimental and Molecular Pathology 2009; 87: 204–211.
7. Fenard D, Lambeau G, Valentin E, Lefebvre J, Lazdunski M, and Doglio A. Secreted phospholipases A₂, a new class of HIV inhibitors that block virus entry into host cells.The Journal of Clinical Investigation 1999; 104: 611–618.
8. Gomes A, Bhattacharjee P, Mishra R, et al. Anticancer potential of animal venoms and toxins. Indian J Exp Biol 2010; 48: 93-103.
9. Ramos O H P and Selistre-De-Araujo H S. Snake venom metalloproteases—structure and function of catalytic and disintegrin domains. Comparative Biochemistry and Physiology C 2006; 142: 328–346
10. Ferrer ED .Snake venom: The pain and potential of the venom. The cold blooded news, TheNewsletter of the Colorado Herpetological Society 2001; 28( 3)
11. DeWys W D, Kwaan H C, and Bathina S. Effect of defibrination on tumor growth and response to chemotherapy.Cancer Research 1976; 36: 3584–3587.
12. Pawelek PD, Cheah J, Coulombe R, et al. The structure of L-amino acid oxidase reveals the substrate trajectory into an enantiomerically conserved active site. The EMBO J 2000; 19: 4204-15.
13. Lee ML, Chung I, Fung SY, Kanthimathi MS, Tan NH. Anti-proliferative activity of king cobra (Ophiophagus Hannah) venom L-amino acid oxidase. Basic and Clinical Pharmacology and Toxicology. 2014; 114: 336–343.
14. Lee ML, Tan NH, Fung SY, Shamala DS. Antibacterial action of a heat-stable form of L-amino acid oxidase isolated from king cobra (Ophiophagus hannah) venom. Comp Biochem Physiol C. 2011; 153: 237-242.
15. Rodrigues RS, Izidoro LF, de Oliveira RJ, et al. Snake venom phospholipases A2: a new class of antitumor agents. Protein and Peptide Letter 2009; 16: 894-8.
16. Lakshminarayanan R, Valiyaveetti S, Rao VS and Kini RM. Purification, characterization and in vitro mineralization studies of a novel goose eggshell matrix protein, ansocalcin. J of Biol. Chemistry 2003 ; 278(5): 2928-2936
17. Shimuta K, Ohnishi M, Iyoda S, Gotoh N, Koizumi N, Watanahe H. The hemolytic and cytolytic activities of Serratia marcescens phospholipase A (PhlA) depend on lysophospholipid production by PhlA. BMC Microbiology 2009 ; 9: 261
18. Lariviere W.R, Melzack, R. The bee venom test: a new tonic-pain test. Pain1996; 66: 271–277.
19. Park HJ, Lee SH, Son DJ et al. Antiarthritic effect of bee venom: inhibition of inflammation mediator generation by suppression of NF-kappaB through interaction with the p50 subunit. Arthritis Rheum. 2004; 50(11):3504-15.
20. Park JH, Jeong YJ, Park KK et al. Melittin suppresses PMA-induced tumor cell invasion by inhibiting NF-kappaB and AP-1-dependent MMP-9 expression. Mol Cells. 2010; 29(2): 209-15.
21. Park MH, Choi MS, Kwak DH et al. Anticancer effect of bee venom in prostate cancer cells through activation of caspase pathway viainactivation of NF-Κβ. Prostate. 2011; 71(8):801-12.
22. Liu S, Yu M, He Y et al. Melittin prevents liver cancer cell metastasis through inhibition of the Rac1-dependent pathway. Hepatology 2008; 47(6): 1964-73.
23. Jang MH, Shin MC, Lim S et al. Bee venom induces apoptosis and inhibits expression of cyclooxygenase-2 mRNA in human lung cancer cell line NCI-H1299. J Pharmacol Sci. 2003; 91(2): 95-104.
24. G and  Garaj-Vrhovac V. Melittin: A lytic peptide with anticancer properties. Environ Toxicol Pharmacol. 2013; 36(2):697-705.

25. Alley M C, Scudiro D A, Monks A, Hursey M L, Czerwinski M J,Fine D L, et al. Feasibility of drug screening with panels of human cell lines using a microculture tetrazolium assay. Can Res 1988; 48:589-601.
26. Marone M. Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sample. Biol Proced Online 2001; 3(1): 19–25.
27. Mansouri M , Mirzaei SA , Lage H , Mousavi SS , Elahian F. The cell cycle arrest and the anti-invasive effects of nitrogen-containing bisphosphonates are not mediated by DBF4 in breast cancer cells. Biochimie 2014; 99: 71-76.
28. Deepika Jan, Sudhir Kumar. Snake Venom: A Potent Anticancer Agent. Asian Pacific Journal of Cancer Prevention 2012; 13:4855-4860
29. Tang N, Xie Q, Wang X, et al. Inhibition of invasion and metastasis of MHCC97H cells by expression of snake venom cystatin through reduction of proteinases activity and epithelial-mesenchymal transition. Archives of Pharmacal Res 2011; 34: 781-9.
30. Feofanov AV, Sharonov GV, Dubinnyi MA, Astapova MV, Kudelina IA, et al. comparative study of structure and activity of cytotoxins from venom of the cobras Naja oxiana, Naja kaouthia, and Naja haje. Biochemistry (Mosc) 2004; 69: 1148-1157.
31. Gallacci M, Cavalcante WLG. Understanding the in vitroneuromuscular activity of snake venom Lys49 phospholipase A2homologues. Toxicon 2010; 55: 1-11.
32. Marcussi S, Sant’Ana CD, Oliveira CZ, Rueda AQ, Menaldo DL, Beleboni RO, et al. Snake venom phospholipase A2 inhibitors: medicinal chemistry and therapeutic potential. Cur Topics Med Chem 2007; 7: 743-756.
33. Gutierrez JM, Alexandra R, Escalante T, Díaz C. Hemorrhage induced by snake venom metalloproteinases: biochemical and biophysical mechanisms involved in microvessel damage. Toxicon 2005; 45: 997-1011.
34. Marshall DM. Enzyme activities and biological functions ofsnake venoms. Appl Herpetol 2005; 2: 109-123.
35. Aguilar I, Guerrero B, Salazar AM, Giron ME, Perez JC, Sanchez EE, et al. Individual venom variability in the South American rattlesnake Crotalus durissus Toxicon 2007; 50:214-224.
36. Naumann GB, Silva LF, Silva L, Faria G, Richardson M, Evangelista K. Cytotoxicity and inhibition of platelet aggregation caused by an L-amino acid oxidase from Bothrops leucurus Biochim Biophys Acta 2011; 1810: 683-694.
37. Alyan MS, Shalaby MA, El-Sanousi AA, Fahmy A, El-Sayed M, Shebl RI. Antiviral and Anticancer Potentials of Snake and ScorpionVenom Derivatives. Inventi Rapid: Molecular Pharmacology 2014; 2014 (2):1-11
38. Lee ML, Fung SY, Chung I, Pailoor J, Cheah SH, Tan NH. King Cobra (Ophiophagus hannah) Venom L-Amino Acid Oxidase Induces Apoptosis in PC-3 Cells and Suppresses PC-3 Solid Tumor Growth in a TumorXenograft Mouse Model. J. Med. Sci. 2014; 11(6): 593-601
39. Liu X, Chen D, Xie L, Zhang R. Effect of honey bee venom on proliferation of K1735M2 mouse melanoma cells in vitro and growth of murine B16 melanomas in vivo. J. Pharm. Pharmacol 2002; 54: 1083–1089.
40. Jang, MH, Shin MC, Lim S, Han SM, Park HJ, Shin I. Bee venom induces apoptosis and inhibits expression of cyclooxygenase-2 mRNA in human lung cancer cell line NCI-H1299. J. Pharmacol Sci 2003; 91:95–104.
41. Hu H, Chen D, Li Y, Zhang X. Effect of polypeptides in bee venom on growth inhibition and apoptosis induction of the human hepatoma cell line SMMC-7721 in-vitro and Balb/c nude mice in vivo. J. Pharm. Pharmacol 2006; 58: 83–89.
42. Moon D O, Park SY, Heo MS, Kim KC, Park C, Ko WS, Choi YH, Kim GY. Key regulators in bee venom-induced apoptosis are Bcl-2 and caspase-3 in human leukemic U937cells through down regulation of ERK and Akt. Int. Immunopharmacol 2006; 6: 1796–1807.
43. Ip SW, Liao SS, Lin SY, Lin JP, Yang JS, Lin ML, Chen GW, Lu HF, Lin MW, Han SM, Chung JG. The role of mitochondria in bee venom-induced apoptosis in human breast cancer MCF7 cells. In Vivo 2008a; 22: 237–245.
44. Ip SW, Wei HC, Lin JP, Kuo HM, Liu KC, Hsu SC,Yang JS , MeiDueyang CTH, Han SM, Chung JG. Bee venom induced cell cycle arrest and apoptosis in human cervical epidermoid carcinoma Ca Ski cells. Anticancer Res 2008b; 28: 833–842.
45. Maher, S, McClean S. Melittin exhibits necrotic cytotoxicity in gastrointestinal cells which is attenuated by cholesterol. Biochem. Pharmacol 2008; 75:1104–1114.
46. Ko SC and Song HS. Inhibitory Effect of Bee Venom Toxin on the Growth of Cervix Cancer C33A Cells via Death Receptor Expression and Apoptosis. The Acupuncture 2014; 31 (2): 75-85
47. Jang DM and Song SH. Inhibitory Effects of Bee Venom on Growth of A549 lung cancer cells via induction of death receptors. Journal of Korean Acupuncture & Moxibustion Medicine Society 2013; 30 (1) : 57-70
48. Keun Young Hur and Ho Sueb Song Inhibitory Effect of Bee Venom Toxin on Lung Cancer NCI H460 Cells Growth through Induction of Apoptosis via Death Receptor Expressions. The Acupuncture Vol. 31 No. 1;2014 : 121-130
49. Liu S, Yu M, He Y, Xiao L, Wang F, Song C, Sun S, Ling C, and Xu Z. Melittine prevents liver cancer metastasis through inhibition of the Rac1- dependent pathway. HEPATOLOGY 2008; 47(6): 1964-1973.
50. Holle, L, Song W, Holle E, Wei Y, Wagner T, Yu X. A matrix metalloproteinase 2 cleavable melittin/avidin conjugate specifically targets tumor cells in vitro and in vivo. Int. J. Oncol 2003; 22: 93–98.
51. Son DJ, Lee JW, Lee YH, Song HS, Lee CK, Hong JT. Therapeutic application of anti-arthritis, pain-releasing, and anti-cancer effects of bee venom and its constituent compounds. Pharmacol. Ther 2007; 115:246–270.
52. Soman NR, Baldwin SL, Hu G, Marsh JN, Lanza GM, Heuser JE, Arbeit JM, Wickline SA, Schlesinger PH. Molecularly targeted nanocarriers deliver the cytolytic peptide melittin specifically to tumor cells in mice, reducing tumor growth. J. Clin. Invest 2009; 119: 2830–2842.
53. Pan H, Soman NR, Schlesinger PH, Lanza GM, Wickline SA. Cytolytic peptide nanoparticles (‘NanoBees’) for cancer therapy. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol 2011; 3:318–327
54. Zhu HG, Tayeh I, Israel L, Castagna M. Different susceptibility of lung cell lines to inhibitors of tumor promotion and inducers of differentiation. J. Biol. Regul. Homeost 1991; 5: 52–58.
55. Raibekas AA and Massey V. Primary structure of the snake venom L-amino acid oxidase shows high homology with the mouse B cell interleukin 4-induced Fig1 protein. Biochem Biophys Res Commun 1998; 248 (3):476-8.
56. Kim KT and Song HS. Inhibitory Effect of Snake Venom Toxin on Colorectal Cancer HCT116 Cells Growth through Induction of Intrinsic or Extrinsic Apoptosis. Journal of Korean Acupuncture & Moxibustion Medicine Society 2013; 30 (1): 43-55.
57. Shim YS and Song HS. Effect of Snake Venom Toxin on Inhibition of Colorectal Cancer HT29 Cells Growth via Death Receptors Mediated Apoptosis. The Acupuncture 2014; 31 (2) : 87-98
58. Yang ZL, Ke YQ, Xu RX, Peng P. Melittin inhibits proliferation and induces apoptosis of malignant human glioma cells. Nan Fang Yi Ke Da Xue Xue Bao 2007; 27:1775–1777.
59. Chen YQ, Zhu ZA, Hao YQ, Dai KR, Zhang C. Effect of melittin on apoptosis and necrosis of U2 OS cells. Zhong Xi Yi Jie He Xue Bao 2004; 2: 208–209.
60. Li B, Gu W, Zhang C, Huang XQ, Han KQ, Ling CQ. Growth arrest and apoptosis of the human hepatocellular carcinoma cell line BEL-7402 induced by melittin. Onkologie 2006; 29:367–371.
61. Zhang C, Li B, Lu SQ, Li Y, Su YH, Ling CQ. Effects of melittin on expressions of mitochondria membrane protein7A6, cell apoptosis-related gene products Fas and Fas ligand in hepatocarcinoma cells. Zhong Xi Yi Jie He Xue Bao 2007; 5:559–563.
62. Moon DO, Park SY, Choi YH, Kim ND, Lee C, Kim GY. Melittin induces Bcl-2 and caspase-3-dependent apoptosis through downregulation of Akt phosphorylation in human leukemic U937 cells. Toxicon 2008; 51:112–120.
63. Lipps BV. Novel snake venom proteins cytolytic to cancer cells in vitro and in vivo systems. J Venom Anim Toxins 1999; 5: 173 183.
64. Gajskia G, Čimbora-Zovkob T, Rakb S, Rožmanc M, Osmakb M and Garaj-Vrhovaca Combined antitumor effects of bee venom and cisplatin on human cervical and laryngeal carcinoma cells and their drug resistant sublines. J. Appl. Toxicol 2014;34: 1332–1341
65. Hui L, Leung K, Chen HM. The combined effects of antibacterial peptide cecropin A and anti-cancer agents on leukemia cells. Anticancer Res, 2002; 22:2811–2816.

66. Wang C, Chen T, Zhang N, Yang M, Li B, Lu X, Cao X,and Ling C. Melittin, a Major Component of Bee Venom, Sensitizes Human Hepatocellular Carcinoma Cells to Tumor Necrosis Factor-related Apoptosis-inducing Ligand (TRAIL)-induced Apoptosis by Activating CaMKII-TAK1-JNK/p38 and Inhibiting. THE Journal of Biological Chemistry 2009; 284(6): 3804–3813.
    

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    How to cite this article:

    El Sharkawi FZ, Saleh SS and Mohamed El Sayed AF: Potential Anti Cancer Activity of Snake Venom, Bee Venom and Their Components in Liver and Breast Carcinoma. Int J Pharm Sci Res 2015; 6(8): 3224-35.doi: 10.13040/IJPSR.0975-8232.6(8).3224-35.

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