Squalene Selectively Protects Mouse Bone Marrow Progenitors Against Cisplatin and Carboplatin-Induced Cytotoxicity In Vivo Without Protecting Tumor Growth^,

Abstract

Squalene, an isoprenoid antioxidant is a potential cytoprotective agent against chemotherapy-induced toxicity. We have previously published that squalene protects light-density bone marrow cells against cis-diamminedichloroplatinum( II) (cisplatin)-induced toxicity without protecting tumor cells in vitro. Here, we developed an in vivo mouse model of cisplatin and cis-diammine (cyclobutane-1,1-dicarboxylato) platinum(II) (carboplatin)-induced toxicity to further investigate squalene-mediated LD-BM cytoprotection including the molecular mechanism behind selective cytoprotection. We found that squalene significantly reduced the body weight loss of cisplatin and carboplatin-treated mice. Light-density bone marrow cells from squalene-treated mice exhibited improved formation of hematopoietic colonies (colony-forming unit-granulocyte macrophage). Furthermore, squalene also protected mesenchymal stem cell colonies (colony-forming unit-fibroblast) from cisplatin and carboplatin-induced toxicity. Squalene-induced protection was associated with decreased reactive oxygen species and increased levels of glutathione and glutathione peroxidase/glutathione-S-transferase. Importantly, squalene did not protect neuroblastoma, small cell carcinoma, or medulloblastoma xenografts against cisplatin-induced toxicity. These results suggest that squalene is a potential candidate for future development as a cytoprotective agent against chemotherapeutic toxicity.

Introduction

Myelosuppression is a major toxicity for most chemotherapy regimens. Myelotoxicity is associated with morbidity, mortality, cost, and, most importantly, with reduced chemotherapy dose intensity and treatment failure [1,2]. The reduction in dose intensity may compromise treatment outcome including disease control and survival in patients with curable malignancies [1]. Among the most commonly used myelosuppressive drugs are the platinum derivatives cis-diammine (cyclobutane-1,1-dicarboxylato) platinum(II) (carboplatin) and cis-diamminedichloroplatinum(II) (cisplatin) [3]. Carboplatin is a second-generation platinum complex having substantial myelosuppressive effects. The toxicity is cumulative in nature and can occur in 18% to 25% of cases [3]. Treatment with high-dose carboplatin (>1200 mg/m²) leads to myelosuppression in >90% of cases [4]. Cisplatin is the first generation platinum compound having mild myelo-suppressive effects (5–6% cases). The toxicity is correlated with peak levels of the drug in the first 2 weeks of treatment [4,5], and its incidence and severity dramatically increase in patients under dialysis (25–100% cases) [5,6] and when used in combination with carboplatin [7].

One of the known mechanisms of toxicity of platinum drugs such as cisplatin and carboplatin is due to cross-linking with nucleic acids and proteins. Both cisplatin and carboplatin are platinum(II) complexes with two ammonia groups in the cis position. Cisplatin has two chloride groups, which are replaced by water molecules in an intracellular aquation reaction. The reaction is driven by the high concentration of water and low concentration of chloride in the tissues. The aquated platinum complex can then react with a variety of macromolecules including RNA, DNA, and protein. The cytotoxicity of cisplatin is correlated closely with platinum DNA interstrand bifunctional N-7 adducts at d(GpG) and d(Apg) [8]. However, so far, the types of DNA lesions responsible for the cytotoxicity of cisplatin have not been clearly established [3]. Other toxic effects of cisplatin in vitro-cultured cell growth includes attenuation of mitochondrial function and the release of reactive oxygen species in the cells [9,10]. Carboplatin has a similar mechanism of action like cisplatin [3].

The mechanism of platinum-induced myelosuppression is not clearly known. Evans et al. [11] reported that after a single dose of cisplatin treatment (4–20 mg/kg) in hybrid mice (C57BL x BALB/c), the light-density bone marrow (LD-BM)-derived colony-forming units (CFUs) were depleted significantly. Nowrousian and Schmidt [12] found similar results of the depletion of the CFUs suggesting that cisplatin may target the hematopoietic stem cell fraction leading to myelosuppression. We previously reported that cisplatin treatment exerts significant toxicity on the hematopoietic stem cell fraction in vitro [13]. Carboplatin has also been found to target the hematopoietic stem cell fraction both in vitro and in vivo [3,14,15]. The mechanism of platinum-induced toxicity on hematopoietic stem cells may be related to its DNA cross-linking and generation of oxidative stress products such as malondialdehyde [16]. Carboplatin may have a similar mechanism of oxidative stress induction as cisplatin [3], and it has been found to decrease glutathione (GSH) level in rat kidney [17] and bone marrow (BM) cells [18]. Antioxidants GSH and metallothioneins are found to prevent cisplatin and carboplatin-induced toxicity [19–22]. Hence, the clinical use of antioxidants have been suggested to reduce cisplatin and carboplatin-induced myelosuppression [21,23–28].

Squalene is an isoprenoid antioxidant that is secreted in human sebum, where it may protect skin from UV radiation [29]. Dietary squalene has been found to have radioprotective activity [30] and exerts anticarcinogenic activity against several compounds by enhancing cellular antioxidant status [31–34]. Our in vitro studies in squalene-mediated cytoprotection indicate that squalene (12.5–25 µM) has selective cytoprotective activity; it protected BM colonies from cisplatin-induced toxicity without protecting neuroblastoma colonies [13]. Importantly, the cytoprotective activity of squalene was equivalent to GSH, a major intracellular antioxidant and detoxifying agent [20]. Furthermore, squalene may have antitumor activity. It has been previously shown that squalene inhibited murine sarcoma growth and survival in a mouse model [35–38]. We found that squalene inhibits the in vitro growth of the NBL-S neuroblastoma cell line [13].

Such differential normal tissue protective, anticarcinogenic, and antitumor activities make squalene a potential cytoprotective agent against chemotherapeutic-induced myelotoxicity [13,29]. To test this possibility, we investigated the in vivo protective activity of squalene in a mouse model of platinum-induced myelotoxicity. We also investigated the potential protective activity of squalene against platinum-induced toxicity against tumor growth in vivo.

Materials and Methods

Cell Culture

Fresh normal mouse BM specimens were obtained from mice; normal LD-BM mononuclear cells (LD-BM cells) were separated by the Percoll method as described [39]. Separated LD-BM cells were immediately used for the CFU assay as described below. Tumor cell lines, SK-N-BE(2) (a neuroblastoma cell line), H-146 (a small cell carcinoma cell line), and D-283 (a medulloblastoma cell line), were obtained from the American Type Culture Corporation (ATCC, Manassas, VA) and maintained according to ATCC guidelines.

Reagents and Drugs

Cisplatin, carboplatin, and reduced GSH were obtained from Sigma-Aldrich, Oakville, ON, Canada. Unless stated otherwise, tissue culture and other reagents/drugs were obtained from Sigma-Aldrich. Both cisplatin and carboplatin were dissolved in normal saline.

Squalene Preparation

Squalene (Squalene iP6, derived from dogfish shark oil) was a gift from Isshogenki International, Hong Kong. Gas chromatography analysis performed in our laboratory established the purity of Squalene iP6 as more than 99.3% [13]. Squalene was mixed with commercially available Intralipid 200-mg/ml fat emulsion to make a 0.8-mg/ml solution of squalene in Intralipid as described [40]. The squalene-Intralipid solution was injected intraperitoneally (i.p.) at 100 mg/kg (1 ml of 2-mg/ml squalene-Intralipid solution injected i.p. into a 20-g BALB/c mouse).

Animals

Eight- to ten-week-old female BALB/c/nude/nude mice were obtained from Charles River Laboratories (Wilmington, MA). Protocols for animal experimentation were approved by the Animal Safety Committee, Research Institute, The Hospital for Sick Children, Toronto, Canada. The details are described in the Supplementary Methods section.

In Vivo Xenograft Assay

The neuroblastoma cell lines SK-N-BE(2), SH-SY5Y, small cell carcinoma cell line H-146, and medulloblastoma cell line D-283 were used for the xenograft study. Briefly, trypan blue-excluded viable cells (6 x 10⁶) were mixed with Matrigel (50 µl of cell suspension in 50 µl of Matrigel) and injected subcutaneously into both flanks of female nude mice (BALB/c, nude/nude; n = 6). The tumor size was measured with a caliper on a biweekly basis, and tumor volume was determined using the formula 0.5ab², where b is the smaller of the two perpendicular diameters as described [41]. As required, tumors were harvested, weighed, and then dissociated into cell suspensions by a simple method of trituration (finely minced tumor tissue transferred to a 15-ml tube and vigorously pipetted for 1–2 minutes). The resulting cell suspensions were centrifuged and washed with PBS twice and then trypsinized in 2 x trypsin EDTA solutions for 15 minutes. This suspension was washed and counted for viability using the Trypan blue assay and was plated in methylcellulose medium for clonogenic study. To study the long-term effect of squalene cytoprotection against tumor growth, 10-week-old BALB/c nude/nude mice (weight, 19.3 g, 95% confidence interval, 19–19.7 g) were used. Group 1 mice were killed when the tumor reached 2 cm³ in size. At the appropriate time, platinum drug was injected i.p. 3 hours after the injection of squalene i.p., and tumor growth was monitored. Mice were killed at the ninth week, and BM cells were isolated to perform the CFUs and CFU-F assays. For the SH-SY5Y cell line, a separate protocol was used as described in the text.

Measurement of BM Protection: CFU Assay

For the evaluation of BM toxicity, we used a method developed by Treskes et al. [42], where LD-BM cells from the mouse BM were isolated, and in vitro colony assay was performed. The assay was performed using a standardized methylcellulose assay system [13] using Methocult GF H4434 (StemCell Technologies Inc., Vancouver, BC, Canada) containing recombinant growth factors including granulocyte colony-stimulating factor (CSF) and erythropoietin. Briefly, normal LD-BM cells were immediately plated in methylcellulose medium (Methocult GF H4434), and CFUs were counted after 14 days.

Measurement of Mesenchymal Stem Cell Protection: CFU-F Assay

The assay was performed using the Mesencult medium (#05501 and #05502; StemCell Technologies) as per the manufacturer's instruction. Briefly, total BM cells were collected from a mouse femur (∼2 x 10⁷ cells) into 2 ml of Iscove's modified Dulbecco's medium supplemented with 2% FBS, and after cell counting, 1 x 10⁶ cells were plated per well in a six-well plate in 2 ml of Mesencult medium. At day 14, colonies were fixed with methanol, stained with Giemsa, and CFU-F (CFU-fibroblast) was counted [43]. Osteogenic and adipogenic differentiation was performed as described [44]. Briefly, BM cells were cultured to three to four passages in Mesencult media and then incubated in osteogenic and adipogenic medium for 3 weeks, and medium was changed every 72 hours. Osteogenic medium contained DMEM, 10% FBS, 0.1 µM dexamethasone, 10 mM glycerophosphate, and 5 µg/ml ascorbic acid. After 3 weeks, cultures were washed with PBS, fixed with 10% formalin, and stained with Alizarin red for calcium deposition and alkaline phosphatase activity. The adipogenic medium was made of DMEM, 10% FBS, 0.1 µM dexamethasone, and 5 µg/ml insulin. After 3 weeks, cultures were fixed with 10% formalin and stained with oil red O stain.

Measurement of Cellular Proliferation and Toxicity by Clonogenic Assay

The assay was performed as previously described [13,45]. A total of 2 x 10³ tumor cells were seeded in methylcellulose medium (Methocult M3134; StemCell Technologies) with 10% FBS with or without treatment with cisplatin, and after 2 weeks of culture at 37°C and 5% CO₂, colonies were counted as described [13].

Measurement of Antioxidant Enzymes

Superoxide dismutase (SOD), glutathione-S-transferase (GST), and glutathione peroxidase (GSPx) were measured using standard assay kits (Cayman Chemical Company, Ann Arbor, MI). For sample preparation, a modified method was used where cell pellets were homogenized in a common lysis buffer (50 mM Tris-HCl, pH 7.6, containing 5 mM EDTA and 1 mM DTT). Briefly, 2 x lysis buffer was added, and samples were homogenized for 5 to 10 seconds on ice and then stored at -80°C (stored for 3–6 months). Subsequently, samples were thawed, protein levels were measured, and the samples diluted in a standard buffer (50 mM Tris-HCl, pH 7.6, 5 mM EDTA); these were pipetted into 96-well plates with additional reagents provided in the kit to measure SOD, GST, and GSPx level. The measurement was done by reading the change in absorbance using a spectrophotometer according to the manufacturer's instructions.

GSH Measurement

A GSH assay kit was obtained from Oxford Biomedical Research, Oxford, MI. Murine BM cells or xenograft tissues were dissociated using a lysis buffer containing 50 mM sodium phosphate, pH 6.7, and 1 mM EDTA. A part of the lysate was used to measure protein concentration by BCA protein assay kit (Pierce, Rockford, IL), and 100 µl of lysates were dissolved in an equal volume of 5% metaphosphoric acid. Glutathione was measured by a colorimetric-based method using a methyl-sulfate reagent (Oxford Biomedical Research). In the assay, GSH present in the lysate reacts with the methyl-sulfate reagent to produce a GSH-thioether, which is converted to chromophoric thione (absorbance, 400 nm) under alkaline conditions (pH > 13.4). A standard curve was obtained using known GSH concentrations. Data were normalized per milligram of protein.

Measurement of Intracellular ROS

The intracellular level of ROS was measured with 2′,7′-dichlorofluorescein diacetate (DCFH-DA; Sigma-Aldrich) as described [46]. Briefly, BM and tumor cells were plated in a 96-well plate (4 x 10⁴ per well) and preincubated with DCFH-DA dye, and the relative fluorescence unit was obtained after 30 minutes in a Gemini Spectra MAX microplate reader (Molecular Devices, Sunnyvale, CA).

Measurement of Tissue Squalene

Spanggord et al. [47] described a hexane-based extraction method to measure squalene, where a reverse-phase HPLC-UV detection method was used. We have used this HPLC method with modification to measure squalene in mouse plasma and found that squalene level in plasma peaked 3.5 hours after i.p. injection [48]. Here, we used the HPLC method to measure squalene level in BM at 3, 24, and 120 hours after a bolus dose of squalene-Intralipid (100 mg/kg) injection. Briefly, after squalene injection, at an appropriate time interval, blood was collected by intracardiac route and then animals were killed to obtain BM. Subsequently, squalene was extracted from plasma, BM, and SK-N-BE(2) tumor xenografts using the hexanebased extraction method as described (details are given in the Supplementary Methods section) [47,48]. A Shimadzu HPLC system (Shimadzu Corporation; Kyoto, Japan) was used. Separation of squalene was achieved within 6 minutes on a 15-cm reversed-phase C₈ analytical column with a particle size of 5 µm using a methanol mobile phase [47,48]. Squalene level was expressed as micrograms per milliliter for plasma and micrograms per gram for BM and xenograft tissues (Supplementary Methods).

Statistical Analysis

Measurements of colony growth of both BM cells and tumor cells were normalized to the percentage of the control and analyzed by one-way ANOVA. The data are presented as mean ± SD. The statistical calculations were performed with GraphPad Prism 4.0 (Hearne Scientific Software, Chicago, IL). A value of P ≤ .05 was considered statistically significant.

Results

Squalene Exerts Protection Against Platinum-Induced Weight Loss

The timing and dose of a cytoprotectant are important factors to obtain effective cytoprotection [23]. It is therefore important to know the pharmacokinetic profile of a cytoprotective agent before undertaking the in vivo experiments. We have used a modified HPLC method [47,48] to measure squalene level in mouse plasma. We found that 3 hours after i.p. injection of a single-bolus 100-mg/kg squalene dose, BM squalene levels reached 25 µg/g per milliliter (Figure 1A; P = .002). This BM squalene level is above the required dose of squalene for BM cytoprotection [13]. Furthermore, we found that the BM squalene level remains above baseline level even at 24 hours and 5 days after the injection of squalene (Figure 1A; P = .0048 and P = .0329, respectively). We also measured the squalene level in the SK-N-BE(2)-derived xenografts after i.p. injection of squalene and found that squalene level increased significantly in the xenograft tissue. However, 24 hours after injection, the squalene level did not remain significantly higher than the baseline level (Figure 1A; P = .1145). Furthermore, the baseline squalene level dropped by 34% (P = .277). Although the differences between these data are not significant, it may be suggestive of the rapid metabolism of squalene in the xenograft tissue.

Figure 1.

Injection of dietary squalene increases tissue level of squalene and reduces platinum-induced weight loss. (A) Histogram shows the level of squalene in BM and plasma of BALB/C nude mice after injection of 100 mg/kg squalene-Intralipid i.p. injection. Squalene level significantly increased in plasma, BM, and xenograft tissues 3 hours after injection. Note that BM squalene level remained significantly higher than baseline level 24 hours after the injection. (B) (i) Histogram showing > 10% weight loss after cisplatin (12 and 15 mg/kg) and carboplatin (100 mg/kg). Squalene (100 mg/kg) administration did not reduce weight. (ii) Squalene significantly reduced cisplatin and carboplatin-induced weight loss. *P < .01, **P < .01, ***P < .0001. #P = .02.

We then screened for the effect of a single dose of cisplatin or carboplatin on weight loss. Groups of mice (n = 8) were injected i.p. with 10 to 15 mg/kg cisplatin or 100 to 120 mg/kg carboplatin, and weights were measured daily. The 12- and 15-mg/kg cisplatin groups and 120-mg/kg carboplatin group showed more than 10% weight loss by day 5, whereas the 10-mg/kg cisplatin and 100-mg/kg carboplatin groups showed less than 10% weight loss by day 5 (Figure 1B). To investigate squalene-induced cytoprotection, drugs were injected 3 hours after the i.p. injection of squalene to SK-N-BE(2) xenograft-bearing nude mice. Using this experimental design, we first demonstrate that squalene treatment significantly reduced both cisplatin (12 mg/kg)- and carboplatin (120 mg/kg)-induced weight loss (Figure 1B), thus suggesting that the timing and dose of squalene injection was appropriate to investigate the potential cytoprotective activity of squalene against platinum-induced BM toxicity.

Squalene Protects Against Platinum-Induced CFUs Toxicity

Initially, we investigated the drug-induced toxicity to CFUs. We found that the 12- and 15-mg/kg cisplatin doses significantly reduced CFUs number (Figure 2A; P < .05). We also measured the white blood cell (WBC) count, which did not show significant changes at the 12- and 15-mg/kg doses (data not shown). Conversely, carboplatin at a 120 mg/kg dose produced significant CFUs toxicity (Figure 2A) and a decrease in white blood count (data not shown). Earlier, we found that cisplatin treatment significantly reduces large CFU-granulocyte macrophage (CFU-GM) colonies in vitro, whereas concomitant treatment with squalene protects the large colonies [13]. Here, we found an equivalent significant protection of large-size CFU-GM colonies (Figure 2B). Similar squalene-induced protection of large CFU-GM was observed in the carboplatin-treated group (data not shown).

Figure 2.

Squalene-induced protection against platinum-induced CFUs toxicity. (A) Histogram showing percent decrease in CFUs after cisplatin and carboplatin treatment. Squalene treatment reversed the decrease in CFUs. Control CFUs colony number was 78.2 ± 8 per 1 x 10⁵ LD-BM cells. (B) Two-week-old CFU-GM colony assay showing the predominance of large-size colonies in squalene- versus cisplatin-treated groups (phase-contrast microscopy, x10). To the right, the histogram shows the quantification of large colonies in squalene versus cisplatin treatment group. Data were converted to percent control of mean value, and comparison was between the platinum alone versus platinum + squalene treatment groups. *P < .01, ***P < .0001.

Squalene Protects Against Platinum-Induced CFU-F Toxicity

Bone marrow microenvironment maintains a pool of mesenchymal stem cells (MSCs) [49]. In vitro cultures established from single-cell suspensions of mouse BM generate colonies of adherent marrow stromal cells, each derived from a single precursor cell (CFU-F) having characteristics of MSCs [50]. Mesenchymal stem cell can differentiate into bone, fat, and cartilage and express Oct-4, a stemness gene involved in embryonic stem cell self-renewal [49]. To establish the CFU-F assay and characterize MSCs, BM cells were collected and grown in special mesenchymal culture medium and colonies were enumerated as described previously [43,50]. When we grew these colonies in the mesenchymal culture medium (detailed in the Materials and Methods section) and subjected to MSC characterization, they expressed Oct-4 and differentiated along osteogenic and lipogenic lineages (Figure 3, A–C). Cisplatin treatment led to dramatic changes in the morphology of CFU-F colonies where normal-appearing CFU-F cells were replaced by flat cells having prominent nucleoli (Figure 3D). Similar morphologic changes were observed in carboplatin treatment group (data not shown). The morphologic changes were associated with a significant reduction in the number of CFU-F colonies. The 12-mg/kg cisplatin dose induced CFU-F toxicity by 38% (P = .037), which was completely reversed by squalene (Figure 3E). The 15-mg/kg cisplatin dose reduced CFU-F number by 65% (P = .006); squalene treatment reversed the loss of CFU-F by 45% (P < .0001). The 120-mg/kg carboplatin dose reduced CFU-F number by 80% (P < .0001) and the addition of squalene reversed the CFU-F loss by 32% (P = .0382; Figure 3E).

Figure 3.

Squalene reduces platinum-induced MSC toxicity. (A, B, and C) Culture and characterization of murine MSCs, which were grown in CFU-F colonies, express Oct-4 and differentiate to osteogenic and lipogenic lineages. (D) A murine MSC colony (CFU-F) showing significant reduction of cellular content after cisplatin treatment. Addition of squalene recovered the cellularity of CFU-Fs. (E) Histogram showing percentage decrease in CFU-F colonies after cisplatin treatment. Squalene treatment reversed the CFU-F decrease. We found 16.6 ± 6 CFU-F colonies per 1 x 10⁶ BM cells. Data were converted to percent control of mean value, and comparison was between the platinum alone versus platinum + squalene treatment groups. *P < .01, ***P < .0001.

Squalene Does Not Protect Tumor Against Platinum-Induced Short-term (5 Days) Toxicity

Above results show that squalene exerted significant protection against platinum-induced BM toxicity in a mice model of SK-N-BE (2) xenograft growth. When the cisplatin-treated xenografts were investigated for the toxicity on the fifth day of treatment, the 15-mg/kg cisplatin group demonstrated a significant reduction in tumor weight (30%; P = .042) and clonogenic activity (63.5%; P = .0147). Cisplatin + squalene treatment reduced the tumor weight by 37% (P = .048) and clonogenic activity by 64.7% (P = .078; Figure W1) suggesting that combination treatment of squalene did not protect the tumor growth from cisplatin-induced toxicity. We found similar results with xenograft from H-146 (a small cell lung cancer cell line) and D-283 (a medulloblastoma cell line), where cisplatin treatment significantly reduced clonogenic growth, whereas squalene treatment had no effect on cisplatin-induced antitumor activity (Figure W1).

Squalene Protects BM Against Long-term Platinum Toxicity Without Protecting Tumor Growth

Considering that platinum drug may exert cumulative toxicity [13], we tested squalene's protective activity against the long-term platinum toxicity in an SK-N-BE(2) xenograft model. In this model, xenograft bearing mice were treated with cisplatin 10 mg/kg once weekly for two consecutive weeks (treatment started at the fifth week when the tumor size reached 0.2–0.25 cm³; n = 4). The 12-mg/kg dose was not used because of the high mortality in mice within the first 2 weeks after injection (data not shown). Carboplatin 100 mg/kg once weekly for two consecutive weeks was used instead of 120 mg/kg for the same reason. Squalene was injected 3 hours before the injection of platinum drugs. At the end of 3 weeks after the last dose of drug (i.e., at the ninth week after tumor inoculation), the animals were killed, and BM was collected. We found that cisplatin treated mice became weak, dehydrated, and lost 12.6% body weight (Figure 4A; P = .0253; n = 6), whereas mice receiving cisplatin + squalene appeared active/alert and showed significant protection against weight loss compared with the cisplatin-treated group (Figure 4A; P = .0323; n = 4). In addition, the skin of cisplatin-treated mice exhibited keratin plaques and appeared dry and wrinkled, whereas squalene-treated mice showed almost no keratin plaques and appeared normal. Furthermore, the cisplatin-treated group showed a 30% reduction in the number of CFU colonies (P = .002) and a 46% reduction of CFU-F colonies (P = .006), whereas the squalene + cisplatin-treated mice showed complete recovery of both CFU and CFU-F colonies (Figure 4, B and C).

Figure 4.

Squalene reduces platinum-induced long-term weight loss and BM toxicity. Histograms showing that squalene treatment reverses platinum-induced (A) body weight loss, (B) CFUs toxicity, and (C) CFU-F toxicity. Note that the cisplatin treated group received drugs for two consecutive weeks, whereas the carboplatin group received drugs for three consecutive weeks. Body weight was taken and BM cell was harvested at the end of ninth week after the inoculation of tumor cells. Squalene was administered 3 hours before each dose of platinum injection. Control CFUs: 82.2 ± 8.6 per 1 x 10⁵ LD-BM cells. Control CFU-F: 17.8 ± 7.2 per 1 x 10⁶ BM cells. Data were converted to percent control of mean value, and comparison was between the platinum alone versus platinum + squalene treatment groups. **P < .001, **P < .0001.

We then investigated the effect of 100 mg/kg carboplatin once weekly for 2 weeks and found that this dose regimen did not significantly reduce mice body weight, whereas an additional dose (100 mg/kg once weekly for three consecutive weeks) led to significant body weight loss (Figure 4A), external signs of toxicity such as dry and wrinkled skin, and a significant reduction of both CFUs (50%; P = .0001) and CFU-F (70%; P = .0036). Addition of squalene showed a dramatic recovery of both CFUs and CFU-F colonies (Figure 4, B and C).

Squalene Does Not Protect Tumors Growth Against the Long-term Platinum-Induced Toxicity

Mice bearing SK-N-BE(2) (0.2–0.25 cm³; n = 4) were treated with squalene (100 mg/kg) once weekly for two consecutive weeks (fifth and sixth weeks after tumor cell inoculation) and were allowed to grow until they reached 2 cm³ in size. Squalene treatment did not affect the xenograft growth significantly (Figure 5A). Cisplatin treatment initially reduced SK-N-BE(2) xenograft size significantly (P = .024) at the seventh week and then regrew (repopulation) at the end of the ninth week (Figure 5A). Carboplatin treatment for the 3-week dose regimen showed similar results of repopulation (data not shown). We recently found that SK-N-BE(2) tumor cell line contain a subpopulation of stem cell-like cells resistant to cisplatin and other chemotherapeutic agents [51]. To test the selective cytoprotective activity of squalene, it is important that the xenograft shows response to drug treatment. We found that cisplatin treatment significantly reduced the clonogenic activity of H-146 and D-283 cells (Figure W1). Considering that methylcellulose-based clonogenic assay measure the clonogenic activity of tumor stem cell fractions [52,53], our data suggest that cisplatin treatment may target the tumor stem cell fraction in these cell lines. We then investigated the effect of cisplatin on the growth of H-146 and D-283 xenografts and found that cisplatin treatment significantly reduced the growth of D-283 (P < .01); squalene treatment increased the cisplatin-induced toxicity, although it was not significant (Figure 5B; P = .074). Although cisplatin showed significant toxicity in the H-146 xenograft growth, the tumor grew rapidly 4 weeks after the cessation of treatment. Squalene treatment did not effect the growth behavior of the xenograft growth after cisplatin treatment (data not shown) suggesting that the H-146 cell line may not be appropriate to investigate the potential protective activity of squalene against platinum-induced tumor growth.We subsequently used a neuroblastoma cell line SH-SY5Y, which is highly sensitive to platinum treatment in vitro (unpublished data). Earlier, we found that squalene did not protect SH-SY5Y cell line from several chemotherapeutic agents including cisplatin and carboplatin in vitro [54]. We developed a new model of in vivo xenograft toxicity, where mice were treated with cisplatin or carboplatin when the tumor size reached 0.05 cm³ in size (the second week of inoculation) instead of allowing the tumor to reach 0.2 to 0.25 cm³ in size before starting the treatment. For the cisplatin group, treatment was administered for two consecutive weeks (the long-term protection regimen for cisplatin; Figure 4) and for three consecutive weeks for the carboplatin group (the long-term protection regimen for carboplatin; Figure 4). When the control (untreated group) reached 2 cm³ in size, all the mice including the treated group were killed, tumor is removed and weighted. The results obtained from this protocol revealed that cisplatin treatment group showed significant reduction in tumor growth (Figure 5C; P = .0024). Most importantly, squalene treatment potentiated the toxicity of cisplatin against SH-SY5Y xenograft growth (Figure 5C; P = .049). In the carboplatin treatment group, significant reduction of tumor volume was observed with the average tumor reaching a size of only 0.2 cm³ compared to >2 cm³ in size of the untreated tumors suggesting significant toxicity. Squalene treatment did not exert any significant influence against the antitumor activity of carboplatin (Figure 5C).

Figure 5.

Squalene does not protect against platinum-induced toxicity to tumor growth. (A) Effect of squalene on cisplatin-induced reduction of SK-N-BE(2) xenograft growth. Treatment started when the tumor volume was 0.2 to 0.5 cm³ in size (second week after tumor inoculation), and tumors were harvested at the end of the ninth week. (B) Significant reduction of D-283 xenograft volume after cisplatin treatment. Note that squalene treatment increased the cisplatin toxicity. Treatment was achieved as explained above. (C) Significant reduction of SH-SY5Y tumor weight after cisplatin (once weekly for 2 weeks) and carboplatin (once weekly for 3 weeks) treatment with and without squalene. Note the significant increase in toxicity in the squalene + cisplatin-treated group. Treatment started when the tumor volume was 0.05 to 0.1 cm³ in size (second week after tumor inoculation), and tumors were harvested at the end of the ninth week when the control tumor reached >2 cm³ in size. *P < .05, **P < .001, ***P < .0001.

Squalene Treatment Selectively Modulates Cisplatin-Induced Reduction of Antioxidant Status in BM Versus Tumor Cells

Earlier, we proposed that the mechanism of squalene-mediated protection of BM cells may involve modulation of the cellular antioxidant system including GSH [13]. Here, we investigated the potential mechanism of squalene-induced cytoprotection using cisplatin-induced BM cell toxicity as a model.

Cisplatin treatment has been found to increase the generation of oxygen free radicals, such as hydrogen peroxide, superoxide anions, hydroxyl radicals, and nitric oxide in kidney and liver cells of rat and mouse [55–57]. Cisplatin reacts with intracellular GSH to form a GS-platinum complex, which reduces the cellular GSH level significantly [58]. Miyajima et al. [59] showed that cisplatin treatment increases ROS levels as detected by DCFH-DA assay and reduces GSH levels in bladder cancer cells. Martins et al. [10] showed that highdose cisplatin led to increased ROS production and GSH depletion in Wistar rats. However, the effect of cisplatin on ROS production and antioxidant status in BM progenitor cells has not been studied. Earlier, we found that 2-µM cisplatin treatment of human LD-BM cells in vitro led to apoptosis, which can be rescued by treating with 25 µM squalene [13]. Here, we investigated the status of ROS and GSH levels in the LD-BM cells after 2-µM cisplatin treatment in vitro for 24 hours and 15-mg/kg cisplatin treatment in vivo for 5 days. When LD-BM cells were treated with 2 µM cisplatin in vitro, ROS level increased (P = .0024) and GSH level decreased. Squalene treatment significantly reduced ROS levels (P = .026) and increased GSH level (P = .041; Figure 6, A and B). The in vivo measurement of GSH supported the in vitro result; GSH level was reduced by more than twofold (P = .006), which was reversed by squalene treatment (P = .012; Figure 6C).

Figure 6.

Squalene reverses cisplatin-induced changes in ROS and GSH levels. (A) Significant up-regulation of ROS production in SK-N-BE(2) and BM cells in vitro. Addition of squalene did not change the ROS level in tumor cells, whereas it reduced the ROS level in BM cells significantly. Note that the basal level of ROS is higher in the tumor cells than in BM cells. (B) Cisplatin treatment significantly decreased GSH level in LD-BM cells, whereas addition of squalene increased BM cell GSH levels significantly compared to cisplatin group in vitro. (C) In vivo data showing similar results as in vitro, where GSH level was restored in LD-BM cells by squalene treatment, whereas tumor GSH level remained unaffected. *P < .05, #P < .01, **P < .001.

We also investigated the effect of squalene on modulating ROS and GSH levels in the tumor cells taking SK-N-BE(2) tumor cell as a model cell line. SK-N-BE(2) cells were treated with 2 µM cisplatin in vitro for 24 hours with and without squalene treatment, and the ROS and GSH levels were measured. The ROS level increased significantly (P = .036) and GSH level decreased marginally (P = .11) after cisplatin treatment. Squalene treatment did not change the status of ROS and GSH significantly (Figure 6, A and B). Interestingly, the basal level of ROS was significantly higher in the SK-N-BE(2) cells compared to BM cells (P = .0503; Figure 6A).

Cisplatin treatment has been reported to reduce antioxidant enzymes SOD, GST, and GSPx activities in liver and kidney leading to toxicity [60]. However, SOD levels in lung and heart remain unchanged after cisplatin treatment [60]. Here, we found that cisplatin treatment increased SOD activity by 93% (P = .007) and reduced GSPx and GST activity by 32% (P = .0502) and 49% (P = .0302), respectively (Figure 7, A–C). Squalene treatment increased the GSPx and GST activities significantly (Figure 7, A and B; P < .02) and reversed cisplatin-induced SOD activity (Figure 7C; P = .005). We also measured the activities of the antioxidant enzymes SOD, GSPx, and GST in the SK-N-BE(2) xenograft derived tissues and did not find significant changes after cisplatin and/or squalene treatment (Figure 7, A–C).

Figure 7.

Effect of squalene on the cisplatin-induced modulation of antioxidant enzyme activities. Cisplatin treatment showed significantly reduced activity of (A) GST and (B) GSPx in the BM cells, whereas (C) SOD activity increased. Such changes were not observed in the SK-N-BE(2) xenograft. Addition of squalene increased GSPx/GST and reversed SOD activity toward normal (untreated group). Squalene treatment did not modulate SOD, GST, and GSPx levels in the tumor cells. After 5 days of treatment with cisplatin, mice were killed, and SOD/GSPx/GST activities were measured in BM cells and in SK-N-BE(2). *P < .05, **P < .001. #P < .02.

Discussion

Platinum drugs are widely used in chemotherapy treatment against cancer. Cisplatin and carboplatin are known to induce myelosuppression, nephropathy, neuropathy, and gastrointestinal dysfunction [10,58–60]. However, the potential effect of cisplatin and carboplatin against BM progenitor cells including the MSC fraction is not known. Here, we show that acute high-dose cisplatin/carboplatin may target the BM progenitor population including the hematopoietic and mesenchymal progenitor fractions. Furthermore, we have shown that squalene, a nontoxic antioxidant found in the diet, and particularly high in olive oil, exerts in vivo protective effect against cisplatin and carboplatin-induced BM toxicity without protecting tumor growth. We also report squalene-mediated differential modulation of the antioxidant system in BM compared with tumor cells after cisplatin treatment.

We used the LD-BM fraction of BALB/c nude mice as representative of primitive BM stem cell fraction. The LD-BM fraction is obtained by Percoll gradient separation and has been consistently shown to be enriched in both short-term and long-term hematopoietic stem cells [61,62]. Earlier, we had shown that cisplatin treatment targets the CFU-GM and CFU-granulocyte-erythroid-macrophagemegakaryocyte fraction of LD-BM cells in vitro [13]. Here, we found that cisplatin may target the LD-BM-derived CFUs in vivo. Furthermore, cisplatin also exerts toxicity against the CFU-F fraction, which is enriched in BM-derived MSCs [50]. Such toxicity against the BM derived stem cells has tremendous clinical implications particularly in the pediatric population where chemotherapeutic drug toxicities may have long-term implications on growth and development. Interestingly, we did not observe a significant drop of WBC count after cisplatin treatment, whereas carboplatin treatment reduced WBC count, which was reversed by squalene treatment (data not shown).

The mechanism of squalene-mediated protection of hematopoietic and mesenchymal progenitor cells from the platinum-induced toxicity is not known. Considering that dietary antioxidant scavenges free radicals including hydroperoxides [63] and that the platinum-induced tissue toxicity may be related to the generation of ROS [16], it may be presumed that squalene may reduce platinum-induced oxidative stress on the BM progenitors. Cisplatin treatment has been found to increase the generation of oxygen free radicals, such as hydrogen peroxide, superoxide anions, hydroxyl radicals, and nitric oxide in kidney and liver cells of rat and mouse [55–57]. Reports obtained from our study indicate that cisplatin treatment increases ROS level in the BM cells as measured by the DCFH-DA assay. The oxidation of DCFH to the fluorescent compound 2′-9′-dichlorofluorescein is modulated mainly by hydroperoxides including H₂O₂ [64] and peroxynitrite [65]. Due to the rapid rate of the dismutation of superoxide anions to H₂O₂, the endogenous H₂O₂ level may be used as an indirect measure of superoxide anion. Our findings that cisplatin treatment increases 2′-9′-dichlorofluorescein fluorescence level in BM cells may reflect cellular oxidant stress because of the increased generation of various free radicals including H₂O₂ and superoxide anions. Several reports suggest that increased ROS generation after cisplatin treatment reduces GSH level and GSPx/GST activity in kidney and liver tissues [55–57,66,67]. Additionally, free radicals may inactivate GSPx through the modification of a cysteine-like essential residues on GSPx [68].

We found that cisplatin treatment significantly reduces GSPx and GST activity in BM cells, whereas SOD level was significantly increased in BM cells. Previous reports suggest significant decrease of SOD in liver and kidney tissues after cisplatin treatment [55,56,66]. We also found that SOD level decreases in a hepatic cell line WRL-68 after cisplatin (2 µM for 24 hours) treatment [69]. Conversely, Sadzuka et al. [60] found that cisplatin treatment did not decrease SOD level in lung and heart, whereas significant reduction in SOD activity was detected in liver tissues suggesting that cisplatin-induced reduction of SOD may be tissue- and cell type-specific. Cetin et al. [70] investigated the level of SOD in patients undergoing high-dose chemotherapy including cisplatin therapy and found that SOD activity was increased significantly in days 3 and 6 after the high-dose chemotherapy. Recent report suggest that MnSOD activity is increased by very high dose cisplatin treatment (500 µM cisplatin for a 1-hour treatment) in a renal cell line, LLC-PK1 [71]. We measured the total SOD activity using Cayman SOD assay kit that measures all three types of SOD (Cu/Zn-, Mn-, and FeSOD). Whether increased SOD activity in the BMcells is due to the increase in MnSOD activity requires further investigation.

Both SOD and GSPx modulate the conversion of highly toxic superoxide anions to molecular oxygen and water in a two-step enzymatic process: first, the dismutation of superoxide anion to hydrogen peroxide by SOD; second, the conversion of hydrogen peroxide to molecular oxygen and water by GSPx and/or catalase. Therefore, the balance between the first and second steps of enzyme activity may be critical to maintain cellular antioxidant defense. Overabundant SOD activity and diminished GSPx activity may lead to a net increase in hydrogen peroxide intermediates. The resulting hydroxyperoxides (OH·) are even more harmful than superoxide anions [72,73] and may be related to several disease pathologies [73–75]. Our data suggest that after cisplatin treatment in vivo, the SOD activity in BM cells increased whereas GSPx activity decreased. The changes in SOD and GSPx activity was associated with reduced GSH and GST levels and a significant decrease in both hematopoietic and MSC colony growth.

Squalene increased the GSPx and GST activity and reduced the cisplatin-mediated increase of SOD activity in BM cells. It is possible that squalene reverses the cisplatin-mediated induction of SOD, thus restoring the balance between SOD and GSPx activity. Mishima et al. [71] found that N-acetyl cysteine (NAC) and cAMP reverses the cisplatin-induced MnSOD activity in LLC-PK1 cell line leading to protection from cisplatin-induced cell death. Considering that squalene treatment reversed the cisplatin-induced SOD activity in BM cells, it will be important to find out whether MnSOD activity is reversed by squalene. Earlier, Sabeena Farvin et al. [76] reported that squalene restored the levels of several antioxidant enzymes including GSPx, SOD, and GST in rat myocardium after isoproterenol-induced myocardial infarction. However, it is not known whether MnSOD or Cu/ZnSOD was increased after squalene treatment. Future studies of squalene-induced SOD modulation should include measuring the activity of all the three major types of SOD (Cu/ZnSOD, MnSOD, and extracellular SOD).

The mechanism of squalene-mediated modulation of SOD level in the LD-BM cells during oxidative stress is not known. Cisplatin treatment may modulate SOD level by activating p38/MAPK signaling pathway [71]. Additionally, Nrf-2 (NF-E2-related factor 2), a transcription factor associated with the restoration of antioxidant system after chemical stress [77], has been found to be activated after cisplatin treatment [78]. Considering that cisplatin may also modulate SOD level by activating Nrf-2 signaling [78], squalene may suppress cisplatin-induced Nrf-2 activity. Therefore, future investigation should take in consideration of understanding the potential link between squalene and the stress signaling pathways including p38/MAPK and Nrf-2. Such investigation may further our understanding of the role of dietary squalene in the regulation of cellular antioxidant balance.

It is crucial that cytoprotective and rescue agents do not protect tumor cells from the toxicity of chemotherapeutic agents. Our study indicates that squalene did not exert a protective effect on tumor cell growth in vivo.We have examined the possible squalene-induced protection of in vivo tumor growth against cisplatin and carboplatin-induced toxicity and found that squalene did not exert a great influence on the tumor growth/toxicity. These data support our previous in vitro data that showed that squalene did not exert protection of neuroblastoma cell lines against platinum-induced toxicity [13]. The mechanism of such selective protection of BM cells versus tumor cell growth by squalene against platinum-induced toxicity is not known. We found that squalene treatment decreased ROS level and increased the GSH level in the BM cells but not in the SK-N-BE(2) tumor cells. It is possible that squalene selectively increases GSH levels in BM versus tumor cells, a phenomenon we earlier described as the “GSH-paradox” [13,79]. We found that OTZ (l-2-oxothiazolidine-4-carboxylate), a GSH prodrug, is able to selectively decrease tumor GSH levels. It was observed that tumor GSH levels are depleted (chemopotentiation) whereas normal tissue GSH levels are elevated (chemoprotection). This phenomenon of a “GSH paradox” suggests that GSH-enhancing agents may be used to differentially protect normal versus tumor tissues [79]. The selective increase of antioxidant system in the BM cells by an antioxidant may be related to the different regulation of antioxidant system in normal versus tumor cells. Genomic instability is a hallmark of tumor cells, and tumor cells may be under a continued environment of oxidative stress, where antioxidant may act as a pro-oxidant [80–82]. Schwartz [83] suggested that dietary antioxidants may act as and antioxidant to normal cells and as a pro-oxidant to cancer cells. The pro-oxidant activity of an antioxidant may be the reason for the antitumor activity of many antioxidants including NAC, lycopene, squalene, and vitamin E [16,28,29, 81,84,85]. Here, we found that the basal level of ROS was significantly higher in the SK-N-BE(2) tumor cells than the LD-BM cells. Whether such a different cellular microenvironment of tumor cells rendered the antioxidant activity of squalene ineffective in the tumor cells needs further investigation.

Another possibility for the selective protection of squalene in BM versus tumor cells may be related to the difference in the cellular uptake or the status of the mevalonate pathway, the latter being the cholesterol synthesis pathway in cells. We have measured tumor squalene and found that, at least in the tumor level, uptake of squalene does not differ. Cancer cells may synthesize its own cholesterol, and therefore, dietary squalene may end up being used up for cholesterol synthesis. This possibility is supported by our findings that squalene level was higher in the tumor xenograft than in BM cells. Furthermore, addition of squalene increased the tumor squalene level by several folds. However, within the next 5 days, the squalene level decreased below the baseline level. It is possible that exogenous squalene may inhibit the cellular synthesis in the cancer cells. This possibility is supported by several preliminary findings that squalene may inhibit the enzyme squalene synthase or HMG-CoA reductase.

Nakagawa et al. [37] found that dietary squalene potentiated the toxicity of several chemotherapeutic agents in murine sarcoma. Earlier, we found that squalene treatment increased the etoposide-induced toxicity of SK-N-SH neuroblastoma cell line in vitro [13]. Dietary squalene may also have direct antitumor activity, which may be related to the inhibition of the HMG-CoA reductase enzyme, the rate-limiting enzyme of the cholesterol metabolism [29,86]. The mevalonate pathway is highly active in tumor cells including mitochondria, and several byproducts of the metabolism are required for protein isoprenylation. Inhibition of the mevalonate pathway by the inhibitor of squalene synthase has been found to reduce tumor growth and potentiate the toxicity of doxorubicin [87,88]. Here, we found that SKN-BE(2) tumor tissue contains a high level of endogenous squalene, which may reflect the high activity of the squalene synthesis pathway in the tumor cells. We also found that addition of exogenous squalene reduced the baseline squalene level in the tumor cells by 34% (P = .277). The administration of squalene to SH-SY5Y xenograft-bearing mice potentiated cisplatin-induced tumor growth reduction. Therefore, it is necessary to investigate whether dietary squalene may exert feedback inhibition to the squalene synthesis pathway in the tumor tissue.

There is an urgent need for the clinical development of safe and nontoxic cytoprotective agents for the adequate management of the adverse effects of cancer chemotherapy [24]. Amifostine represents one of the most well studied and common cytoprotectants in clinical practice. However, intravenous administration of amifostine in a phase III clinical trial resulted in toxicities in 41% of head and neck cancer patients, including hypotension, hypocalcemia, nausea, vomiting, and allergy, and necessitated withdrawal of treatment [89]. The hypotensive property of amifostine precludes treatment of patients on hypertensive medication and requires administration of a large volume of saline administered before amifostine to prevent a major drop in blood pressure [85]. Nonetheless, a substantial drop in blood pressure may occur, leading to discontinuation of amifostine administration [85]. In addition, even subcutaneous injections of amifostine have incited severe reactions including Stevens-Johnson syndrome, erythema multiforme, toxic epidermal necrolysis, and bullae [90]. Rescue agents such as CSFs are also used to ameliorate the toxic effects of chemotherapy and radiotherapy. They primarily function by stimulating the BM to undergo hematopoietic expansion. Colony-stimulating factor such as granulocyte CSF is administered 24 hours after the last dose of chemotherapy or radiotherapy because CSFs administered in conjunction with chemotherapy enhances BM toxicity by increasing neutropenia and thrombocytopenia [91]. Here, we have shown that squalene administered in conjunction with cisplatin and carboplatin protects BM progenitors. Furthermore, squalene treatment reduced cisplatin-induced body weight loss in both short-term (5 days) and long-term (4 weeks) treatment schedules without protecting tumor growth. In a phase I trial, adult males were given 860 mg of squalene daily for 20 weeks to study the cholesterol-lowering effect of squalene. Squalene was found to be safe and tolerable at high doses; of 26 patients studied, only 1 patient complained of diarrhea [92]. Considering the nontoxic nature of dietary squalene, the potential clinical assessment of this agent in the pediatric population is warranted. Squalene, being a nontoxic dietary antioxidant, may be particularly beneficial in the pediatric population against platinum-induced BM progenitor cell toxicity. Storm et al. [30] demonstrated the protective effect of squalene against radiation-induced BM damage. Hamilton et al. [93] found that Hjorth adjuvant (10% w/v squalene in water) increased the survival and proliferation of murine BM-derived cells. Whether squalene-induced protection of BM cells also extends to toxicity related to other chemotherapeutic agents, such as alkylating agents, requires further investigation. Further investigation of squalene cytoprotection against chemotherapeutic agents including using orally administered dose is necessary for future clinical trials in humans.

Although we found that squalene does not protect tumor cell growth from the cisplatin-induced toxicity, it is unknown whether squalene may support the growth of a small subfraction of tumor stem cells. We recently found that SK-N-BE(2) and H-146 cell lines contain a small fraction of tumor stem cell-like cells having a very high tumorigenic capacity [45], whereas the SK-N-BE(2)-derived tumor side population cells are highly resistant to cisplatin treatment [51]. Here, we found that although cisplatin treatment initially led to shrinkage of the SK-N-BE(2) and H-146 (data not shown), the tumor growth quickly regained suggesting the potential role of tumor stem cell fraction in repopulation [94]. Squalene treatment did not significantly affect this repopulation phase. Further investigation is necessary to find out if squalene may reduce the repopulation of tumor stem cell after chemotherapy.

Future work still remains to be done to extend the cytoprotective findings we have reported here. Firstly, it will be necessary to investigate whether squalene can also protect BM cells against chronic platinum-induced toxicity including protection against nephrotoxicity, hepatotoxicity, and ototoxicity. Secondly, the potential BM protective effect of squalene against other frequently administered chemotherapeutic agents such as cyclophosphamide should be explored.

In summary, we found that squalene exerts a cytoprotective effect on BM and against platinum-induced BM toxicity in mice. Considering the nontoxic nature of dietary squalene, the potential use of this agent for the pediatric population undergoing cancer treatments should be considered.

Supplementary Methods

Animals

Eight- to ten-week-old female BALB/c/nude/nude mice were obtained from Charles River Laboratories. All animal experimentation was in accordance with the criteria of the Guide for the Care and Use of Laboratory Animals prepared by the National Academy of Sciences, Washington, DC. Protocols for animal experimentation were approved by the Animal Safety Committee, Research Institute, The Hospital for Sick Children, Toronto. The mice were acclimatized 1 week before the planned experiments. They were fed ad libitum and were housed in individual cages on a 12-hour light/dark cycle. In the initial experiment, mice were dosed with cisplatin (8–15 mg/kg, i.p.; n = 5 for each group). The control mice (n = 5) received saline only. After 5 days of treatment, the mice were weighed and then anesthetized, and blood was removed by intracardiac puncture. The mice were then euthanized by cervical dislocation, and BM was collected as described [1]. Blood was processed for total WBC count using an automated analyzer in the clinical laboratory division of The Hospital for Sick Children. Bone marrow specimens were either processed for CFU and CFU-F assays as described below or stored appropriately for the measurement of GSH, SOD, and GSPx as described below. For the measurement of tissue squalene levels, xenograft-bearing mice were divided into four groups (each group; n = 4). Group 1 received normal saline. Groups 2 to 4 received squalene 100 mg/kg, i.p. Mice from each group were euthanized at 0-, 4-, 24-, and 120-hour intervals. The animal blood, BM, and tumor tissues were collected for the HPLC measurement of squalene (described below). For the measurement of squalene-induced cytoprotection, mice were divided into groups (n = 6) receiving cisplatin (12 or 15 mg/kg i.p.) with or without squalene (100 mg/kg of squalene-Intralipid solution, i.p., 3 hours before the injection of cisplatin) or NAC (10 mg/kg dissolved in normal saline i.p. 1 hour before the injection of cisplatin). The control group received Intralipid solution only.

For the measurement of long-term protection of squalene, a separate protocol was used as described in the Materials and Methods section in the main text.

Squalene Extraction and HPLC Measurement

Briefly, after squalene injection into mice, blood was collected, animals were killed, and BMs and tumor tissues were collected. Blood was collected in a microcentrifuge tube and centrifuged at 8000 rpm, and the serum was stored at -80°C. For squalene extraction, serum was thawed, mixed with propanol and hexane, and vortexed, and the clear top hexane layer was collected in a glass tube and evaporated under a stream of liquid nitrogen. The dried residue was reconstituted in 100 µl of methanol and subjected to HPLC qualification. For measurement of tumors, 50 mg of tissue was first mixed with 0.5 ml of water, homogenized, and kept at room temperature for 5 to 10 minutes for the total lysis of cells. Subsequently, 0.5 ml of propanol was added and homogenized again; then 0.5 ml of hexane was added to the homogenized suspension, and the extraction procedure was completed as described previously. Squalene concentrations in BM cells were measured by collecting the cells in PBS solution and by storing them at -80°C. For the extraction, samples were thawed and centrifuged, and the pellet was weighed. 0.1 ml of water was added, and the solution was incubated at room temperature for 5 to 10 minutes. Then, 0.1 ml of propanol was added per 10 mg of pellet, vortexed vigorously for next 5 minutes, 0.1 ml of hexane was added and vortexed again as per the extraction procedure. Squalene level was expressed as micrograms per milliliter for plasma and micrograms per gram for BM and xenograft tissues.

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Abbreviations

BM

bone marrow

LD-BM

light-density bone marrow

ROS

reactive oxygen species

GSH

glutathione

MSC

mesenchymal stem cell