Anticancer Effects of Medical Malaysian Leech Saliva Extract ( LSE )

The concept of the medical application of leeches can be traced back to the beginning of civilization. Traditionally, in many countries including Malaysia leech application had been used for many human body disorders starting from the conventional usage of leech for bloodletting. Moreover, many reports mentioned the usage of leech in skin diseases, nervous system abnormalities like brain congestion, urinary and reproductive system problems (nephritis, vaginitis). In addition, ocular inflammation, dental problems and hemorrhoids were also treated by leech therapy [1-3].


Introduction
The concept of the medical application of leeches can be traced back to the beginning of civilization. Traditionally, in many countries including Malaysia leech application had been used for many human body disorders starting from the conventional usage of leech for bloodletting. Moreover, many reports mentioned the usage of leech in skin diseases, nervous system abnormalities like brain congestion, urinary and reproductive system problems (nephritis, vaginitis). In addition, ocular inflammation, dental problems and hemorrhoids were also treated by leech therapy [1][2][3].
In the 20 th century, leeches have been a model for many extensive studies to evaluate the usage of leech products, especially leech saliva, for therapeutical purposes [4,5]. Consequently, a large number of peptides and proteins with tremendous clinical applications have been identified and characterized in leech extract, such as antithrombin agents [6,7], antiplatelet compounds [8][9][10], etc. Nowadays, leeches have been introduced as promising tools in microsurgery and reconstructive operations after many reports about better salvage of grafted tissues and amputated digits after leech application [11,12].
With regard to cancer and metastasis therapy, many researchers delineated the effective usage of leech saliva and leech salivary gland extract as an anti-metastatic agent. It has been outlined that salivary gland extract from Haementeria ghilianii and Haementeria officinalis inhibited the metastatic colonization of lung tumor cells which were injected intravenously into the experimental animals [13]. Other research described a booming synthetic hirudin preparation as an efficacious metastasis inhibitor of a wide range of malignant tumor cells, such as pulmonary carcinoma, osteocarcinoma, breast carcinoma, leukemia, etc. [14]. Recently, intensive researches led to the isolation of a protein named ghilanten from the leech H. ghilianii salivary gland extract with factor Xa inhibitory and anti-metastatic activities [15].
Putting in mind that leech saliva was not established for tumor treatment as a cytotoxic agent, we aimed from this research to examine the antiproliferative activity of leech saliva from the medicinal Malaysian leech against human small cell lung cancer cell line SW127 in vitro and to evaluate the effectivity of its combination with known antitumor drugs.

Chemicals, reagents and instrumentation
Leibovitz's L-15 medium, bovine serum albumin and arginine hydrochloride were supplied by Sigma Aldrich. Phosphate buffered saline (PBS, 1X sterile solution) was procured from Amresco. The Cell Culture Company PAA was the manufacturer of L-glutamine (L-Glu, liquid, 200mM), penicillin/streptomycin (pen/strep, 100X), fetal bovine serum FBS mycoplex and accutase ® . CellTiter-Glo ® luminescent cell viability assay was obtained from Promega. Trypan blue dye and sodium chloride were the products of Merck. Amresco was the supplier of Bradford reagent kit. Carboplatin (cis-Diamine [1,1-cyclobutanedicarboxylato]platinum II) was from Calbiochem. Irinotecan hydrochloride (USP reference standard) was the product of Rockville, MD.

Leech sampling and saliva collection
Leeches, Hirudinaria manillensis (Lesson 1842), were collected from Cheneh Lake, located in Trengganu, Malaysia. They were maintained in well-aerated plastic containers filled with un-chlorinated tap water. Water was regularly changed every two days. The collected leeches were kept under 12 h: 12 h light and dark cycle at the room temperature (25 ± 1°C). Samples of the collected leeches were sent to Leeches Biopharm (UK) Ltd, for taxonomy and identification.
Leech saliva extract (LSE) was collected from the starved leeches as we already described using 0.011 M arginine in normal saline as a phagostimulatory solution [16].
Only the colorless salivary fluids vomited by the fed leeches were pooled and centrifuged at +4°C and at 2500 rpm for 10 min, and the supernatant was filtered using 0.45 µm Sartorius ® filter paper. The resultant LSE was aliquoted in 1 ml glass tubes and lyophilized for 24 h. The lyophilized LSE was dissolved in a proper volume of distilled water to be concentrated ten folds, and the resultant solution was termed as ten-time concentrated leech saliva extract (10 × LSE) which was used during the experimental procedures after filtration using a sterile 0.2 µm Sartorius ® filter paper.

Total protein estimation
Total protein assay was performed according to the standard protocols of Bradford assay [17] and Amresco Bradford reagent kit using BSA as a standard protein and the phagostimulatory solution as a blank.

Cancer cell line and cell culture
Human small cell lung cancer SW1271 cell line was obtained from the American Type Cell Collection ATCC. Cells were cultivated at an initial inoculums cell concentration of 10 4 cells/cm 2 in 15 ml complete growth media (CGM) which consists of Leibovitz's L-15 medium supplemented with 10% FBS (v/v), 0.3 g/L of L-Glu, and 1% (v/v) pen/ strep in Corning ® 75 cm 2 canted neck cell culture flask. The cultivated cells were incubated at 37°C in CO 2 -free humidified atmosphere. Media was changed as needed. When the monolayer of anchorage-dependent cell line is near 90% confluent, they were subcultured using accutase ® as dissociating agent [18]. Cell counting was performed using trypan blue dye exclusion method [19].

The antiproliferative activity of LSE
Cells were seeded at a density of 10 4 cells/wellin a sterile Corning Costar ® 96-well flat bottom cell culture microplate containing 200 µl of CGM using 8-channel eppendrof ® micropipettor. After all, the microplates were incubated at 37°C in free-CO 2 humidified environment for 24 hours [18]. After 24-hour incubation period; the medium was discarded and replaced by new 180 µl of CGM. After that, volumes of 20 µl, a series of double dilution of the sterile 10 × LSE were pipetted.
Other plates were prepared following the same protocols replacing 10 × LSE by Carboplatin and Irinotecan as positive controls and the ten-time concentrated phagostimulatory solution 10 × PHS as a vehicle control with serial twofold dilution of each. Another negative control plate was prepared containing untreated cells (10 4 cells/well) cultivated in 200 µl of CGM.
In addition, two plates were prepared using 20 µl of series of double dilution of mixtures consisting of 10 µl of 10 × LSE mixed with 10 µl of 100 µM Carboplatin or 10 µl of 100 µM Irinotecan.Finally, all plates were incubated at 37°C in free-CO 2 humidified atmosphere for 5 days.

In-vitro cytotoxicity assay and dose-responsive curves
The antiproliferative effect of LSE was performed using CellTiter-Glo ® luminescent cell viability assay based on measuring the luminescence signal resulted from the reaction between the Ultra-Glo TM recombinant luciferase and The ATP molecules produced by the metabolically viable cells in the presence of Mg +2 and molecular oxygen. CellTiter-Glo assay was fulfilled according to the standard protocols provided by the manufacturer [20]. The percentage inhibition was calculated from the following equation: The concentration of the test sample which inhibits 50% of cell growth (IC 50 ) was averaged from three replicates and estimated from plotting the percentage of cell growth inhibition against test sample concentration [21]. Plots were carried out using Four Parametric Logistic Equation using Sigma Plot 11.0 software.

LSE collection and total protein estimation
A total volume of 45 ml (pH 6.39) of LSE was collected and lyophilized. Prior to the experiment, the lyophilized LSE was dissolved in 4.5 ml distilled water. The total protein estimation exhibited that the collected LSE contained a total protein of 62.549 ± 3.840 µg/ml. Consequently, it was assumed that the initial concentration used during the cytotoxic activity was 625.49 µg/ml because LSE was concentrated ten folds (10 × LSE) after freeze-drying.

The cytotoxic activity of LSE
Results exhibited that LSE had a remarkable anti-proliferation activity against human small cell lung cancer (SW1271 cell line). The concentration of the total protein of leech saliva extract that inhibits the growth of 50% of the treated cells after 5 days of incubation (IC 50 ) was 119.844 µg/ml ( Figure 1). On the other hand, the cytotoxic effect of leech  saliva extract was compared with two other reference standard drugs, Irinotecan and Carboplatin, which are currently in use as anticancer agents. It was found that the IC 50 of these agents were 5.813 µg/ml and 18.754 µg/ml, respectively ( Figure 1). Furthermore, findings of the experiment showed that the ten folds concentrated phagostimulatory solution (10 × PHS) had no effects on cell proliferation.

LSE in combination with Irinotecan and Carboplatin
Furthermore, findings exhibited that there is a kind synergism between the cytotoxicity activity of LSE and that of the experimental reference standards, Irinotecan and Carboplatin. The concentrations of LSE that can supress 50% of cell growth when combined with another anticancer agent (IC 50comb ) were calculated from the plots. It was found that LSE has IC 50comb of 51.463 µg/ml when combined with Irinotecan which is approximately 57.1% less than the IC 50 of LSE used alone ( Figure 2 and Table 1). However, Irinotecan experienced about 11.5% decline in the IC 50 value to be 5.146 µg/ml. On the other hand, roughly 4.6% reduction in IC 50 of LSE was observed when combined with Carboplatin to have an IC 50comb of 114.261 µg/ml. On the contrary, Carboplatin showed a dramatic decline in IC 50 value by 65%, meaning that IC 50comb of carboplatin when combined with LSE was 6.449 µg/ml ( Figure 3 and Table 1).

Discussion The cytotoxic activity of LSE
Our findings revealed that leech saliva extract of the medicinal Malaysian leech H. manillensis possessed a dose-dependent cytotoxic effect against small cell lung cancer cell line SW1271. The total protein concentration of LSE that could inhibit 50% of cell growth was 119.844 µg/ml. The maximum percentage inhibition (60%) was observed at a total protein concentration of 625.490 µg/ml corresponding to the tentime concentrated LSE, which clarifies the reason of concentrating the extract by ten folds before performing the experiment, where as protein concentrations less than 40 µg/ml did not exhibit a remarkable antiproliferation activity, corresponding to the fifth dilution.
As far as we know, we are the first who demonstrated the cytotoxic activity of medicinal leech salivary gland secretion, at least against small cell lung cancer. The previously published data evidenced the antimetastatic activity of leech extract from Haementeria ghilianii and Haementeria officinalis which had been referred to the anticoagulant factors existed in these extracts [13,22,23]. Other reports outlined the usage of the antithrombin peptide, hirudin, from H. medicinalis as a metastatic inhibitor against a wide range of cancers [13,14]. On the other hand, natural products and their synthetic derivatives currently play a leading role in cancer therapy. The sources of natural anticancer include plants, animals, microorganisms and marine creatures [24]. From annelids, several antitumor components were identified from the body tissue and coelomic fluids of the earthworm Eisenia fetida. It was reported that the coelomic fluids of earthworm caused HeLa cell growth inhibition in a dose dependent manner with a concentration of 1 mg/ml was able to inhibit 84% of cell growth [25].
Other studies delineated that the antimicrobial and antitumor activities are related to each other. The first anti-tumoural antibacterial compound was discovered in 1940 from Actinomyces antibioticus and then named actinomycin D [24,26]. Additionally, some proteins extracted from the annelid earthworm exhibited both cytotoxic and antimicrobial effects besides to their hemolysis activity [27,28]. Thus,   we can suggest that the antiproliferative activity of LSE maybe due to the existence of antimicrobial components which we previously evidenced [16].
Nowadays, the controversial role of antioxidants in cancer prevention has been arisen from many reports which linked lower cancer incidence with higher consuming of antioxidants-containing diets, such as fruits and vegetables [29]. Many reports outlined that natural antioxidants may display this prophylactic activity via many suggested mechanisms, like the prevention of free radicals-induced DNA damage, cellular components destruction, etc. [30]. Considering all, the current research revealed that the medicinal Malaysian leech saliva extract possessed both antioxidant (not published data) and cytotoxic activities which make it as a promising tool in cancer therapy and prophylaxis.
Furthermore, Irinotecan which we used in our research as a reference standard drug showed a more potent cytotoxic activity than LSE against SW1271 cell line. Irinotecan growth inhibitory activity was dose-dependent with IC 50 of 5.813 µg/ml. It was found that a concentration of 62.315 µg/ml resulted in about 95% cell growth inhibition and a concentration of about 2 µg/ml was able to induce a considerable percentage inhibition of approximately 30%. Dose responsive curve of Irinotecan became less important after the seventh dilution. Irinotecan is the drug of choice for lung cancer patients. SN-38 is the active metabolite of Irinotecan in vitro. It was reported that IC 50 values of SN-38 ranged from 1-300 nM for many lung cancer cell lines (PC-9, PC-9/ZD, PC-14, SBC-3, a549, H69 and PC-7) [31]. Whereas, we demonstrated that the IC 50 of Irinotecan against small cell lung cancer was about 5.813 µg/ml, corresponding to about 1000 nM.
In addition, we also established that Carboplatin was more potent than LSE as an anti-proliferative agent but still less cytotoxic than Irinotecan. Dose responsive curve displayed IC 50 of 18.754 µg/ ml. The maximum cell growth inhibitory activity was observed at a concentration of 37.130 µg/ml and then activity was not significant after the fifth dilution. Carboplatin is the typical cytotoxic agent of the second generation of the platinum-based anticancer drugs [32]. Many derivative of Carboplatin was prepared and tested against a wide range of cancer cell lines. The IC 50 of carboplatin and its synthetic derivatives ranged from 5-50 µM against many human cancer cell lines (HOS, MCF7, A2780, A2780cis, etc.) [33]. Likewise, we found that the IC 50 of Carboplatin on SW1271 was 18.754 µg/ml, corresponding to about 50 µM. This wide difference between the cytotoxic activity of LSE and the two reference standard drugs can be logically referred to the fact that we used in our investigation the crude LSE which contains, according to what we already published, a wide range of peptides and proteins [34].

LSE in combination with Irinotecan and Carboplatin
We found that the concurrent application, in vitro, of LSE and Carboplatin showed a synergistic cytotoxic effect on small cell lung cancer because of a 65% reduction in the IC 50 value of Carboplatin, whereas, LSE did not experience a considerable decline in IC 50 when combined with Carboplatin. On the other hand, we evidenced a kind of supra-addition between LSE and Irinotecan when cells were treated with both agents simultaneously. The IC 50 of LSE was reduced by 57% reaching the value of 51.463 µg/ml and the IC 50 of Irinotecan showed 11.5% down-fall to be 5.146 µg/ml. In general, many theories have been suggested to explain the interactions between two active drugs including the following [35] including: • Multiple, concurrent, or complementary effects on a single enzyme.
• Alterations in drug uptake.
• Enhanced drug activity or decreased drug inactivity.
The synergism between LSE, Carboplatin and Irinotecan could be a result of one or more of the above mentioned mechanisms. Thus, more studies are needed to reveal the mechanism of action of LSE as antitumor agent. Moreover, this difference in combination effectivity might be referred to the mechanism of action of each compound and to the stage of cell cycle at which each agent functions [31]. For example, some researchers reported an antagonistic effect of the sequential administration of gefitinib and irinotecan. They argued that gefitinib administration would result in a rise in the G0-G1 phase and a decline in S phase populations. The decreased S phase population was not sensitive to irinotecan. On the contrary, treating cells with SN-38 followed by gefitinib exhibited synergistic effects. They reported that SN-38 treatment produced S phase population increase which is sensitive to irinotecan [31].
It was reported that chemotherapy of cancer by multiple drugs would produce better survival and therapeutic efficacy than the results obtained by using a single agent. Combination chemotherapy may also induce maximum cell kill and boarder spectrum of cytotoxicity [35]. Furthermore, many studies had outlined some acute toxic events (nausea, vomiting, etc.) and delayed toxicity (alopecia, myelosuppression, etc.) accompanied chemotherapy regimens by Irinotecan and Carboplatin [36]. On the contrary, literature about leech therapy did not mention serious cytotoxic conditions except for some post-leeching infections which can be handled by the conventional antibiotics [37]. Consequently, we aimed from this experiment to evaluate the capability of LSE to enhance the cytotoxic effect of the previously known anticancer agents, Irinotecan and Carboplatin, at lower doses. Lower doses would result in less adverse effects of these agents. Based on our research result, we recommend using leech or its salivary gland secretion as an adjuvant therapy, especially when using Carboplatin.

Conclusion
Aside from the well-known antimetastatic activity of leech saliva, we reported here for the first time that the salivary gland secretion obtained from the medicinal Malaysian leech, H. manillensis, had an antiproliferative activity against small cell lung cancer besides to its positive synergism with Carboplatin. Hence, further studies still are needed on this issue to isolate and identify the active principle, to study the mechanism of action, to evaluate its effect on other cell line types, etc.