Background
Melanoma, a dreaded form of skin cancer, affects deeper skin layers of the body and spreads rapidly to other tissues and organs[
1]. Chronic exposure of skin to sunlight or UV radiation is a major risk factor for the development of melanoma characterized by alterations in the synthesis of melanin pigment[
2,
3]. Chemotherapeutic drugs, radiation and immunotherapy have been widely used to treat melanoma[
4,
5]. In spite of these options, the median survival rate of melanoma patients is approximately 6 months and hardly 5% of these patients may survive upto 5 years[
6]. Tamoxifen (TAM) is a key member of the selective estrogen receptor modulator family used for the treatment of breast cancer, glioma, cholangiocarcinoma, ovarian cancer and leukemia[
7‐
11]. Tamoxifen inhibits estrogen receptor (ER), although it may exert its effect in ER independent manner too. While the expression of ER in melanoma remains ambiguous, the anticancer effect of tamoxifen has been studied in various melanoma cells[
12‐
14]. Tamoxifen in- combination with other drugs has shown marginal success in combating melanoma[
15,
16]. Failures are attributed to the development of resistance due to limited drug distribution within the tumor cells. Therefore, we hypothesized that increase in the levels of drugs into tumor cells may eventually enhance the therapeutic index.
Plasma membrane contains nanometer-sized dynamic microdomains enriched in cholesterol, sphingolipids and gangliosides. These microdomain structures are integral to the regulation of influx or efflux of drugs. Depletion of membrane cholesterol disrupts integrity of lipid rafts and concurrently enhances the permeability of ions and small non-electrolytes[
17,
18]. Among various cholesterol depleting agents available, methyl-β-cyclodextrin (MCD), a highly water soluble cyclic heptasaccharide consisting of β-[
1‐
4] glucopyranose unit, is the most effective agent for depletion of cholesterol from the cells[
19,
20]. We and others have shown that MCD or its modified forms enhance the cytotoxic effect of various chemotherapeutic drugs[
21‐
23].
We demonstrate that in comparison to tamoxifen alone, MCD treatment enhances the sensitivity of cells towards tamoxifen and thus establishes a promising new strategy for improvement in the outcome of chemotherapy.
Discussion
Due to accompanying drug resistance, effective therapeutic option to treat melanoma is still a medical challenge and nearly no improvement has been made for past thirty years. In this study, we investigated the growth inhibitory effect of tamoxifen in-combination with MCD
in vitro and in isografted mice model of melanoma. We have demonstrated for the first time that only tamoxifen in-combination with MCD was effective in inhibiting the proliferation of melanoma cells regardless of Ras-Raf mutation status (Figure
1).
Tumor recurrence and metastases due to activating mutation in the mitogen-activated kinase (MAPK) pathway attributed to high mortality rate in melanoma patients. Mutation in MAPK enrolls enzyme Ras and Raf signalling cascade that leads to oncogenic cell proliferation and escape from apoptosis[
4]. While human melanoma cell line (A375) carries BRAFV600E mutation and murine melanoma cell line (B16F10) accommodate distinct patterns of mutation in Ras gene with no active mutation in B-Raf oncogene[
26]. Therefore, both human and murine melanoma cell lines were used to address the therapeutic outcome of tamoxifen in-combination with MCD. These mutational events are linked to activation of ERK and Akt and the high occurrence of deregulation pathways thus providing a rationale for the development of target based chemotherapeutics for the treatment of melanoma[
27]. Although, medical fraternity aims to formulate therapeutic strategies toward targeting mutated B-Raf/N-Ras, its downstream molecules ERK and Akt have not been tested[
28,
29]. Recently, the introductions of BRAF inhibitors and new immunotherapies have provided more efficient treatment options with negligible toxicity[
30].
Studies have shown that various signalling molecules enriched on the lipid rafts of plasma membrane are associated with a number of biological processes and their disruption impairs these signaling events[
31,
32]. Hence, various components of plasma membrane have been a promising target for cancer chemotherapy. Cholesterol, an important component of lipid rafts, maintains the stability and architecture of cell membrane. Its accumulation has been reported in cancers such as prostate and oral cancer[
33,
34]. Also, cholesterol metabolism is highly dysregulated in cancers including myeloid leukemia and breast cancer[
35,
36]. Amount of cholesterol present in the lipid rafts of cell membrane influences trafficking of drugs and other molecules through diffusion or receptor meditated uptake[
17]. In these contexts, cholesterol depleting agents, cyclodextrins, are gaining importance in pharmaceutical industries because of their effectiveness in enhancing the bioavailability and solubility of drugs[
37,
38]. MCD is a frequently used FDA approved cyclodextrin to disrupt lipid raft. Depletion of cholesterol from the plasma membrane by MCD leads to modification of membrane permeability thereby altering the signalling and the transport of many molecules in cells[
39‐
42]. Also, it is been reported to cause apoptosis and caveolae internalization in addition to abrogation of Akt signaling in human epidermoid carcinoma cells[
43]. Present study provides evidences that depletion of cholesterol from the membrane sensitizes cells towards tamoxifen mediated cell death through down regulation of Cav-1 and reduced phosphorylation of Akt/ERK. Additionally, we show that MCD not only potentiated the cytotoxic effects of tamoxifen in melanoma cells
in vitro (Figure
1) but also in B16F10 isografted mice model (Figure
4).
Tamoxifen alone or in-combination with other chemotherapeutic drugs has been reported to show poor response rate in the treatment of melanoma[
15,
16]. In this study, MCD was used as a tool to enhance the cytotoxic effect of tamoxifen, and we found that this combination treatment synergistically inhibited the proliferation of melanoma cells.
In vitro combination of tamoxifen and MCD efficiently induced DNA fragmentation and arrest in G1 phase of cell cycle (Figure
2) in A375 and B16F10 cells.
In vivo, we demonstrated that combination of tamoxifen (20 mg/kg) and MCD (64 mg/kg) significantly suppresses the tumor growth of mice as compared to either agent alone with no apparent body weight loss and toxicity to vital organs. The reduction in tumor size is primarily due to enhanced antiproliferative and antiangiogenic effect of combination treatment. Our results are in agreement to the previous report wherein the decrease in the expression of PCNA and CD31 has been correlated with reduction of tumor size and overall disease free survival[
44]. Quantitation of tamoxifen in tumor samples by mass spectrometry highlights the fact that reduction of tumor size was essentially achieved because of increase in tamoxifen levels in the tumor of mice administered with tamoxifen and MCD combintaion as compared to tamoxifen alone (Figure
5C). Concomitantly, the increased drug uptake specifically in tumor reduces the accumulation of tamoxifen in liver and kidney and thus it is likely to be less toxic to the organs, which is a major concern of chemotherapy (Figure
5D, E).
Akt and ERK are major regulators of cell survival pathways governed by growth factors and various cytokines[
45]. Activation of PI3K/Akt or ERK pathways are associated with cell differentiation, cell proliferation and resistance to apoptosis in various cancers including melanoma[
46‐
48]. Cav-1 is an important player for the regulation of cellular cholesterol homeostasis, a process that controls the accumulation of cholesterol on cell membrane. In Cav-1 knock-out mice, depleted level of free cholesterol on the surface of mouse embryonic fibroblast and mouse peritoneal macrophages has been reported[
49]. In an earlier study, we have reported a positive correlation between Cav-1 levels to rapid progression of melanoma in mice fed with high fat diet[
50]. Cav-1 is known to exert direct or indirect effect on the activation of Akt and ERK pathways[
24,
25]. Thus, it is likely that decrease in Cav-1 levels acts as an upstream event in deactivation of intrinsic Akt and ERK growth stimulatory signals in highly metastatic melanoma cells (A375 and B16F10) whereas in non-metastatic melanoma cells (B16F1) no such effect was detected (Figure
6B, C, Additional file
3: Figure S2D respectively). Our findings indicate that cholesterol supplementation prevented MCD potentiated tamoxifen cell death
in vitro as well as
in vivo (Figures
3 and
4) by reversing the molecular alterations as shown in schematic model (Figure
6F).
Material and methods
Drugs, chemicals and antibodies
Tamoxifen (TAM), methyl β-cyclodextrin (MCD), cholesterol (CH) and methylthioazole-tetrazolium (MTT) were purchased from Sigma-Aldrich (Sigma Aldrich, MO). TAM and MCD were dissolved in ethanol and water respectively to prepare 100 mM stock and further diluted in culture medium. Antibodies against pAkt, Akt, pERK, ERK, Cav-1, PARP, Bax, Bcl-2, Cdk4, Cdk1/2, cyclin D1, pRb, Rb, ERα, ERβ and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Cell culture conditions
A375 (human melanoma), B16F10 (murine melanoma) and B16F1 (non metastatic melanoma), HeLa (cervical) and MCF-7 (breast) cancer cells were purchased from American Type Culture Collection (ATCC) Manassas, VA and maintained in our in-house Cell Repository. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% heat inactivated FBS (Hyclone, UT), Penicillin (100U/ml), Streptomycin (100 μg/ml) (Invitrogen Life Technologies, CA) and incubated at 37°C in 5% CO2 incubator (Thermo Scientific, NC).
Mode of MCD and tamoxifen combination treatment
Cells were pre-exposed to MCD for 4 h. Thereafter, cells were washed with fresh medium and then medium containing indicated concentrations of tamoxifen was added for 24 h. For cholesterol (CH) treatment, cells were incubated with MCD for 4 h, washed thereafter and fresh medium containing cholesterol (100 μg/ml) was added. Four hours later, tamoxifen was added and cells were incubated for further 24 h.
MTT (methylthioazole tetrazolium) assay
Cells (5 × 10
3/well) were plated in 96-well plates and allowed to adhere for 24 h at 37°C. Next day, cells were treated with varying concentration of tamoxifen for 24 h with or without MCD or cholesterol as described earlier in mode of treatment. Viability of cells was measured by MTT assay as described[
50]. Any synergistic effect resulting from combination of drugs was calculated by coefficient of drug interaction (CDI) as follows:
AB is the ratio of the combination of drug groups to control group; A or B is the ratio of the single drug group to control group. The CDI value > 1: antagonistic effect, CDI = 1: additive and CDI < 1: synergistic effect[
51].
Whole cell lysate preparation and western blotting
Whole cell lysates were prepared and immunoblotting was performed as described previously[
50].
Long term clonogenic survival assay
Cells (5 × 103/well) were plated in 12-well plates and allowed to adhere for 24 h. Cells were treated as per experimental requirement. After 24 h of treatment medium was replaced with fresh medium and cells were allowed to grow for 10–15 days. After completion of experiment, surviving cells were washed with PBS and fixed with chilled 3% paraformaldehyde. The surviving cells were stained with 0.05% crystal violet dye and images were captured by camera (Olympus, Tokyo, Japan). The number of colonies was counted by using Image J software.
Cells were treated with MCD and tamoxifen as described earlier. Also, tumor sections of mice were stored at -80°C in TRIzol reagent (Invitrogen, CA) after excision until processing for RT-PCR. Total RNA from the cells and tumors was extracted as per the manufacturer’s instructions (TRIzol reagent Invitrogen, CA). cDNA synthesis and RT-PCR were performed as described earlier[
24]. Primer pairs used are as follows: Cav-1 5′-AGA CTCGGAGGGACATCTCTACAC-3′ (F), 5′-ACTGTGTGTCCCTTCTGGTTCTG-3;(R) and for β-Actin 5′-ATCTGGCACCACACCTTCTACAATGAGCTGCG-3′ (F), 5′-CGTCATACTCCTGCTTGCTGATCCACATCTGC-3′ (R). The annealing temperature used for both Cav-1 and β-actin was 58°C.
Cholesterol estimation
Cells (3 × 105) were plated in 35 mm culture dish and treated with MCD, cholesterol and tamoxifen as described earlier. Cells were lysed in PBS containing 2% Triton X-100 for 10 min. After centrifugation (12,000 rpm, 15 min), resulting supernatant was used for cholesterol estimation. For cholesterol release assay, medium was collected and concentrated by using Speed Vac (Thermo Savant, MA). In animal experiments blood was collected from mice fed with 2% cholesterol by approved tail cap method. Cholesterol was estimated by using kit with sensitivity range of 1 mg/dL–750 mg/dL purchased from Spinreact (Girona, Spain) as per the manufacturer’s instructions.
LDH release assay
Cells (3 × 105) were plated in 35 mm culture dish and treated as described earlier, medium was collected and LDH release was measured according to manufacturer’s protocol using LDH activity assay kit (Spinreact, Girona, Spain).
Cell cycle analysis
Cells (3 × 10
5) were plated in 35 mm culture dish and treated with 2.5 mM of MCD for 4 h, washed twice with fresh medium and further grown in culture medium for additional 12 h. In experiments related to MCD and tamoxifen, cells were treated with both the drugs as described earlier. Cells were collected and processed for cell cycle analysis as described elsewhere[
52].
Agarose gel electrophoresis
Cells (3 × 10
5) were plated in 35 mm culture dish and treated with MCD and tamoxifen as described earlier and agarose gel electrophoresis and LDH release assay performed as described elsewhere[
53].
Mass spectrometric analysis
Tamoxifen was extracted from tumors, plasma of tumor bearing mice and from liver, kidney of mice injected with MCD and tamoxifen combination according to described method[
54]. Tamoxifen was quantified in biological samples in duplicate by selected reaction monitoring using hybrid quadrupole Orbitrap mass analyzer (Q-Exactive, Thermo Scientific, Germany) in a high resolution mode. Briefly, 5 μL of the final extract was injected to Accela UPLC (Thermo Scientific, Germany) and the separation was performed using a reverse phase Hypersil Gold C18 5 μm column (150 × 4.6 mm) with a flow rate of 500 μL/min of binary solvent system consisting of mobile phase A: water with 0.1% formic acid and mobile phase B: acetonitrile with 0.1% formic acid. The mass spectra were acquired in high resolution (70000) mode by using Xcalibur and data was processed by Quant software (Thermo Scientific, Germany). The method consisted of full scans and targeted MS/MS of selected precursor ion at a defined mass and retention time according to described settings[
55]. The standard curve was developed by plotting the log
10 value of area under curve (AUC) of the selected fragment of tamoxifen (m/z 72.0808) by extracted ion chromatogram (XIC) against log
10 value of serial dilutions of pure tamoxifen ranging from 50 fg to 500 ng. Tamoxifen was identified and quantified by comparing the XIC of selected fragment ion (m/z 72.0808).
In vivo experiments
All animal experiments were performed according to the Institutional guidelines, following a protocol approved by the Institutional Animal Ethic Committee (IAEC). Five to six weeks old male C57BL/6J (weight 20 ± 2 g) were acquired from experimental animal facility (EAF) of National Centre for Cell Science, Pune, India. B16F10 cells (1 × 106/mice) were injected subcutaneously on the right flank of each mouse. After 12–14 days, palpable tumor bearing mice were randomly divided into four groups (n = 6). Group (a), mice administered with vehicle control, group (b), mice administered with MCD (64 mg/kg, intraperitoneally), group (c), mice administered with tamoxifen (20 mg/kg, orally) and group (d), mice administered with MCD and tamoxifen. MCD and tamoxifen were dissolved in sterile water and ethanol respectively and further diluted with PBS. In cholesterol feeding experiment, mice were fed with chow containing 2% cholesterol for 30 days and total serum cholesterol was measured every 15 days. Subsequently, mice were divided into two groups. Group one referred as control group whereas group two mice were administered with MCD and tamoxifen. Both the groups of mice were fed with cholesterol during the course of experiment. At the end of experiment, mice were sacrificed by cervical dislocation and tumors excised. Size of tumors during the course of experiment was measured using caliper in two dimensions. Tumor volume (mm3) was calculated according to the formula AXB2X0.52 (A, length; B, width; all parameters in millimeters). For immunohistochemical and histopathological studies, sections of tumors and organs were fixed into 10% paraformaldehyde immediately after excision. Remaining part of tumors was stored at -80°C for RT-PCR and lysate preparation used for Western blotting.
Immunohistochemical and histopathological studies
Fine sections (4 μm) were prepared from formalin fixed paraffin embedded tumor tissue and fixed on glass slides (Safeline Histopathology, Pune, India). For immunohistochemistry, slides were deparaffinized by xylene solution twice for 10 min and subsequently dehydrated in graded alcohol (100%, 95%, 70% and 50%). Endogenous peroxidase activity was blocked by 0.01% H2O2. For antigen retrieval, slides were boiled in sodium citrate buffer (0.01 M, pH 4.5) at 100°C for 10 min and allowed to cool at room temperature. BSA (0.2%) was used for blocking for 1 h. After washing with TBST, slides were probed with CD31 and PCNA antibodies specific for IHC (Santa Cruz Biotechnology, CA) and incubated at 4°C overnight. Slides were washed with TBST and probed with compatible HRP-conjugated secondary antibody for 3 h. Slides were stained with diaminobenzidine (DAB) for 10 min followed by counterstaining with hematoxylin and eosin. Slides were mounted and analysis of indicated proteins was performed. For histopathology, deparaffinized slides were stained with hematoxylin and eosin and microscopic analysis for cell density, cellular morphology and necrosis was performed and images were captured by DP71 camera attached with microscope (Olympus, Tokyo, Japan). The staining of cells was quantified by Image J software.
Statistical analysis
Statistical analysis and data comparison were performed by Student’s 2-tailed unpaired t-test by using Sigma Plot software (Systat Software Inc, CA). The values of P <0.05 were considered statistically significant. Quantitation of colonies and relative staining of PCNA of cells were done by using NIH Image J software (Image J Freeware;
http://rsb.info.nih.gov/ij/).
Acknowledgements
We thank Dr. S.C. Mande, Director, and Dr. G.C. Mishra, former Director, National Centre for Cell Science, for being supportive and giving all the encouragement to carry out this work. Authors thank Dr. Vijayakumar MV, NCCS, Pune, India. Dr. Sandeep Singh, NIBMG, Kalyani, India and Dr. Amrendra Kumar Ajay for critical reading of the manuscript. Support from other group members and all technical staff of NCCS is also duly acknowledged.
Financial supports
This work was supported by intramural funding from NCCS, Department of Biotechnology, Government of India. NM, ASM and BC thank Council for Scientific and Industrial Research, India and PM and SVS thank University Grants Commission, India for providing fellowships.
Note
This work was carried out as part of fulfillment of Ph.D. thesis of NM to be submitted to the Savitribai Phule Pune University, Pune, India. The work was partly presented at first Indian Cancer Congress (ICC), New Delhi, 21–24 November, 2013 and in 5th International Conference on Translational Cancer Research (ICTCR), New Delhi February 6–9, 2014.
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Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
Conceived and designed the experiments: MKB and NM. Performed the experiments: NM, PM, ASM, SVS, and BC. Performed the mass spectrometry experiments: GV and MJK. Analyzed the data: MKB and NM. Wrote the paper: MKB and NM. All authors read and approved the final manuscript.