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Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo
Biochemical and Biophysical Research Communications xxx (2015) 1e7
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Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo* Natalia Girola a, *, Carlos R. Figueiredo a, Camyla F. Farias a, Ricardo A. Azevedo b, Adilson K. Ferreira b, Sarah F. Teixeira b, Tabata M. Capello c, Euder G.A. Martins d, ~o H.G. Lago c Alisson L. Matsuo e, Luiz R. Travassos a, Joa ~o Paulo, SP, Brazil Experimental Oncology Unit (UNONEX), Federal University of Sa ~o Paulo, SP, Brazil Laboratory of Tumor Immunology, Institute of Biomedical Sciences, University of Sa c ~o Paulo, SP, Brazil Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of Sa d ~o Paulo, SP, Brazil Institute of Biosciences, University of Sa e ~o Paulo, SP, Brazil Interdepartmental Group of Health Economics (Grides), Federal University of Sa a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 October 2015 Accepted 8 October 2015 Available online xxx
Natural monoterpenes were isolated from the essential oil of Piper cernuum Vell. (Piperaceae) leaves. The crude oil and the individual monoterpenes were tested for cytotoxicity in human tumor cell lineages and B16F10-Nex2 murine melanoma cells. In the present work we demonstrate the activity of camphene against different cancer cells, with its mechanism of action being investigated in vitro and in vivo in murine melanoma. Camphene induced apoptosis by the intrinsic pathway in melanoma cells mainly by causing endoplasmic reticulum (ER) stress, with release of Ca2þ together with HmgB1 and calreticulin, loss of mitochondrial membrane potential and up regulation of caspase-3 activity. Importantly, camphene exerted antitumor activity in vivo by inhibiting subcutaneous tumor growth of highly aggressive melanoma cells in a syngeneic model, suggesting a promising role of this compound in cancer therapy. © 2015 Elsevier Inc. All rights reserved.
Keywords: Camphene Melanoma Apoptosis Antitumor
1. Introduction Malignant melanoma is a most aggressive skin cancer and in its metastatic form, is highly resistant to conventional therapeutic agents. Immunomodulatory antibodies are promising therapeutic agents but may raise adverse collateral effects, therefore, new molecules from natural sources are constantly being studied, representing alternatives for drug development [1e4]. Common anticancer effects of natural products include inhibition of cell proliferation and of signaling pathways, induction of apoptosis, de-
*
Camphene induces intrinsic apoptosis on melanoma cells. * Corresponding author. Unidade de Oncologia Experimental, Departamento de ~o Paulo, Rua Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa ~o Paulo, SP 04023-062, Brazil. Botucatu 862, 8 andar, Sa E-mail address: [email protected] (N. Girola).
stabilization of cytoskeleton integrity, cell cycle modulation, cell migration and metastasis inhibition. These compounds are thus important tools in chemical and pharmacological investigation [3,5,6]. Presently, we characterized the in vitro cytotoxic activity on different tumor cells of the crude essential oil extracted from leaves of Piper cernuum and its monoterpene constituents. Anticancer activities have been attributed to monoterpenes [7], and a-pinene and limonene have been well characterized [8e10], but the antitumor activity of camphene has not been reported before. In this work we observed that camphene induced intrinsic apoptosis in B16F10-Nex2 melanoma cells following the endoplasmic reticulum (ER) stress with liberation of calcium, chromatin and chaperon proteins, as well as caspase-3 activation. A promising antitumor activity was seen in vivo, using a syngeneic melanoma model. All these results suggest that camphene is a candidate for cancertherapy drug development.
http://dx.doi.org/10.1016/j.bbrc.2015.10.041 0006-291X/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: N. Girola, et al., Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/ 10.1016/j.bbrc.2015.10.041
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N. Girola et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
2. Materials and methods 2.1. Plant material and extraction procedures
values in comparison to untreated cells as previously described [13]. Cisplatin was used for comparison of positive toxicity. All experiments were performed in triplicates.
gico do Branches of P. cernuum were collected at Parque Ecolo ~o, Sa ~o Paulo State, Brazil (Latitude: 23 530 4200 Pereque, Cubata South and Longitude: 46 250 3000 West) in July, 2012. For extraction of crude essential oil, fresh branches (200 g) were subjected to steam distillation in a Clevenger type apparatus for 4 h, giving 320 mg of a pale yellow essential oil (yield 0.16%). The oil was dried at room temperature and stored in a sealed vial under refrigeration prior to analysis.
B16F10-Nex2 cells (5 105 cells/well) were cultured on 6-well plates and incubated with camphene at 70 mg/mL at 37 C for 3 h, 4 h and 5 h. DNA was extracted using the TELT buffer (50 mM TriseHCl pH 8.0, Triton X-100 0.4%, 2.5 mM EDTA pH 9.0, and 2.5 M LiCl) and analyzed in Agarose gel electrophoresis as previously described [14].
2.2. Isolation of main compounds from essential oil
2.6. Chromatin condensation analysis
Part of the crude essential oil from branches of P. cernuum (300 mg) was subjected to CC on silica gel eluted with CH2Cl2 and CH2Cl2:MeOH (98:2, 95:5 and 9:1) to afford 67 fractions (6 mL each), which were individually analyzed by FID-GC. These fractions were pooled in ten groups (A1 e A10). Groups A3 (12 mg) and A4 (7 mg) were individually purified by prep. TLC (SiO2 e CH2Cl2: MeOH 99:1) to afford carvacrol (8 mg) and tymol (3 mg), respectively. Group A6 (8 mg) was composed by pure a-terpineol while group A8 (10 mg) was purified by prep. TLC (SiO2 e CH2Cl2: MeOH 98:2) to afford linalol (6 mg). Groups A1 (75 mg) and A2 (52 mg) were pooled and subjected to CC on silica gel soaked with AgNO3 (15%) eluted with CH2Cl2 and CH2Cl2: acetone (99:1 and 98:2) to afford 70 fractions (6 mL each), which were individually analyzed by FID-GC. These fractions were pooled in seven groups (B1 e B7). Groups B3 (3 mg) and B4 (32 mg) were composed by pure a-pinene and camphene. Prep. TLC (SiO2/AgNO3 15% e CH2Cl2:acetone 99:1) separation of group B5 afforded limonene (2 mg) and myrcene (3 mg) while group B7 was composed by pure p-cymene (4 mg). All isolated compounds displayed purity higher than 98% (FID-CG analysis). The identification of the components was based on the comparison of their mass spectra with those in the spectrometer data base (Willey 229 library) and confirmed by determination of their Kovats indexes, which were determined relative to the retention times of a series of n-alkanes [11]. The oils and isolated compounds were stored in DMSO at e 20 C (DMSO, SIGMA).
B16F10-Nex2 cells (1 104) were cultivated overnight on Hi-Q4 dish and incubated with camphene at 70 mg/mL for 5 h. After incubation, cells were stained with 1.25 mM Hoechst 33342 (Invitrogen, Carlsbad, CA) for 10 min, washed two times with PBS 1X and further examined by fluorescence microscopy in a Nikon BioStation IM microscope. Images were processed with ImageJ software (V 1.46r, National Institutes of Health, USA).
2.3. Cell lines and culture conditions The murine melanoma cell line B16F10-Nex2, is a melanotic, aggressive sub-line derived from an original B16F10 melanoma cell line provided by the Ludwig Institute for Cancer Research (LICR), ~o Paulo branch, and was established at the Experimental Sa ~o Paulo (UNIFESP), and Oncology Unit, Federal University of Sa lulas do Rio de Janeiro (BCRJ), reg. deposited in the Banco de Ce 0342. The following human cancer cell lines: melanoma A2058, breast cancer SKBR-3, cervical cancer HeLa, human myeloid leukemia HL-60, were provided by the LICR. The human glioblastoma U87-MG cell line was provided by Prof. O. Keith Okamoto, from ~o Paulo. Cells were cultivated as previously University of Sa described [12]. 2.4. Cell viability assay Crude essential oil from branches of P. cernuum and isolated monoterpenes were incubated with 1 104 tumor and nontumorigenic cells for 24 h at different concentrations ranging from 0 to 100 mg/mL. Cell viability was quantified using MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (SigmaeAldrich, St. Louis, MO, USA), and was quantified as percent
2.5. DNA degradation
2.7. Mitochondrial transmembrane potential (Djm) B16F10-Nex2 cells (1 105) were incubated with 5 nM of tetramethylrhodamine ethyl ester (TMRE, Molecular Probes, OR, USA), a fluorescent probe for intact mitochondrial transmembrane potential, for 30 min at 37 C with 5% CO2 in different 5 mL FACs tubes. Subsequently, 70 mg/mL of camphene, 100 mM of the positive carbonyl cyanide 3-chlorophenylhydrazone (CCCP, SigmaeAldrich, St. Louis, MO, USA), and negative control (RPMI medium) were added into separated cell tubes and incubated at 37 C. CCCP is a well-known protonophore used for positive mitochondrial transmembrane potential disruption [15]. Fluorescence readings were performed after 10 and 30 min of incubation in a FACSCalibur (BD Bioscience, Franklin Lakes, NJ) and further analyzed using the FlowJo software (TreeStar Inc., Ashland, OR, USA). 2.8. Calcium release Release of Ca2þ from ER, upon treatment with camphene was detected using the Fluo 4 NW kit (Invitrogen). B16F10-Nex2 (5 104) cells were seeded in 96-well plates and processed according to the manufacturer's instructions. Then, cells were incubated with or without camphene at 70 mg/mL in the presence or absence of 2 mM of thapsigargin (T) (Enzo Life Science) and fluorescence values were obtained every 60 s, for 1 h, in a plate spectrophotometer (Spectra Max M2e, Molecular Devices). 2.9. Endoplasmic reticulum stress B16F10-Nex2 cells (2 104) were incubated with camphene in four chamber dishes at 70 mg/mL and the ER integrity was assessed by the ER red fluorescent probe ER-Tracker (Life-technologiesTM) following the manufacturer's instructions. After incubation, tumor cells were incubated with ER-Tracker for 10 min at 37 C, and then tumor cells were washed in PBS and immediately observed in a fluorescence microscope (BioStation, Nikon). 2.10. Annexin V assay B16F10-Nex2 cells (5 105) were cultured in 6-well plates and further incubated with camphene 70 mg/mL or complete medium (negative control) for 5 h at 37 C. Phosphatidylserine detection
Please cite this article in press as: N. Girola, et al., Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/ 10.1016/j.bbrc.2015.10.041
N. Girola et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
was performed using the Annexin V-FITC Apoptosis Detection Kit (SigmaeAldrich, St. Louis, MO) as previously described [16]. Quantification of live cells (AV/PI), necrotic cells (AV/PIþ), early apoptotic cells (AVþ/PI) and late apoptotic cells (AVþ/PIþ), was performed using FlowJo software (version 9.5.3, Tree Star Inc., Ashland, OR, USA). 2.11. In vivo subcutaneous melanoma growth Male C57Bl/6 mice were subcutaneously grafted in the flank with 5 104 B16F10-Nex2 cells. After 12 days of tumor challenge, animals were treated with 10 daily peritumoral doses of 300 mg (total 10 mg/kg) of camphene. DMSO (1%) in PBS was used as vehicle control. Mice were euthanized when the maximum allowed volume of 3000 mm3 was achieved. Tumor volume was calculated and analyzed as previously described [12]. Animal experiments were carried out according with the UNIFESP Ethics Committee for Animal Experimentation. 2.12. Cell protein extraction and Western blotting B16F10-Nex2 cells (106) were treated with 35 or 70 mg/mL of camphene for 3 h. After treatment, proteins in cell lysates were analyzed by Western blotting as described elsewhere [17]. The following antibodies were used: anti-b-actin, anti-HmgB1, anticaspase-3 or cleaved caspase-3 and anti-calreticulin, all purchased from Cell Signaling Technology (Beverly, MA, USA). b-Actin was used as loading control. Secondary antibodies conjugated with IgG horseradish peroxidase were purchased from SigmaeAldrich (St. Louis, MO, USA).
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Table 1 IC50 values obtained for crude essential oil from branches of Piper cernuum against different tumor cells in vitro. Lineages
IC50 ± S.D. (mg/mL) Crude essential oil
B16F10-Nex2 A2058 HeLa HL-60 U87-MG
39 24.6 23.6 15.5 19.0
± ± ± ± ±
cisplatin
2.0 1.1 0.1 3.5 0.0
53 43 20.4 21.2 45.0
± ± ± ± ±
4.0 1.5 1.2 1.5 6.0
3.3. Cytotoxic effects of camphene in different tumor and nontumorigenic cell lines The cytotoxic effects of camphene (IC50) were evaluated in tumor and non-tumorigenic cell lines as shown in Table 3. The concentration of 70 mg/mL was therefore used in the subsequent experiments with B16F10-Nex2 melanoma cells. 3.4. Camphene induces apoptotic hallmarks in murine melanoma B16F10-Nex2 cells At 70 mg/mL, camphene induced cell shrinkage and DNA Table 2 IC50 values obtained in B16F10-Nex2 cell for different compounds from essential oil from branches of Piper cernuum and its chemical structures of monoterpenes. Monoterpene
Structural formula
IC50 ± S.D. (mg/mL)
a-pinene
24 ± 0.4
Camphene
70 ± 4.0
Limonene
59 ± 3.0
Carvacrol
82 ± 4.0
Tymol
88 ± 0.7
Myrcene
>100
p-cymene
>100
a-terpineol
>100
Linalol
>100
2.13. Statistical analysis The experiments were performed in triplicate and the values are expressed as means ± standard deviations (S.D.). Student's t test was used for statistical analysis of in vitro and in vivo experiments. For significance analyses GraphPad Prism 4.0 software was used (La Jolla, CA); p < 0.05 was considered a significant difference. 3. Results 3.1. Cytotoxicity of the essential oil from branches of P. cernuum The cytotoxicity of the essential oil from branches of P. cernnum was evaluated in murine and human cancer cells, as shown in Table 1. The IC50 values were obtained and compared with those for cisplatin, taken as a positive control, against the same cells. The crude essential oil was most cytotoxic in HL-60 and U87-MG cell lines, and with the exception of Hela cells was generally more active than cisplatin. 3.2. Natural monoterpenes from P. cernuum and their cytotoxicity against different tumor cells Monoterpenes isolated from P. cernuum were a-pinene, camphene, limonene, carvacrol, tymol, myrcene, p-cymene, aterpineol, and linalol. Their structure formulas and cytotoxicity against B16F10-Nex2 melanoma cells are shown in Table 2. apinene and limonene were the most cytotoxic species that have already been tested in biological systems. Camphene comes next, and its anti-tumor activity has been studied for the first time in the present work.
Please cite this article in press as: N. Girola, et al., Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/ 10.1016/j.bbrc.2015.10.041
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N. Girola et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7 Table 3 IC50 ± S.D. values (mg/mL) for camphene in different cancer cell lines. Lineages
Camphene
B16F10-Nex2 A2058 HeLa HL-60 U87-MG SKBR-3
71.2 35.2 110.1 27.0 55.4 34.7
± ± ± ± ± ±
4.3 1.2 5.1 2.0 1.6 2.5
condensation in tumor cells, with pyknotic nuclei indicated by yellow arrows in Fig. 1A. DNA fragmentation was also evidenced by Agarose gel electrophoresis (Fig. 1B). Camphene also induced significant exposure of phosphatidylserine after 5 h incubation with
melanoma cells, as shown by positive Annexin-V FITC staining, and denoting early apoptosis (Fig. 1C). At the same time half of the cells double stained with Annexin-V and PI indicating membrane damage or late apoptosis as previously described [8]. 3.5. Endoplasmic reticulum (ER) and mitochondrial alterations induced by camphene Camphene caused disturbances in the endoplasmic reticulum and mitochondria of tumor cells, both related to the intrinsic apoptotic process. We observed that camphene induced significant condensation of ER in B16F10-Nex2 cells (80% of positive cells) after treatment with 70 mg/mL, as compared to the negative control (30% of positive cells) (Fig. 2A). This was shown by high fluorescence of cells incubated with the ER-specific red fluorescence probe, ERtracker. We observed also that camphene promoted an increase
Fig. 1. Cell morphology and DNA degradation in camphene-treated melanoma cells. (a) Evaluation of chromatin condensation in camphene-treated cells. B16F10-Nex2 cells were treated with camphene at 70 mg/ml for 5 h and analyzed by fluorescence microscopy (scale bars, 100 mm). (b) Agarose gel electrophoresis of DNA fragmentation in B16F10-Nex2 cells induced by 70 mg/mL camphene at 37 C at different times (3h, 4h and 5 h). (c) Annexin V and PI labeling. B16F10-Nex2 cells were treated with 70 mg/mL of camphene for 5 h and stained with Annexin V-FITC and PI. Analysis was performed by fluorescence microscopy. The values are percentages obtained from three independent experiments. *p < 0.05, compared to negative control.
Please cite this article in press as: N. Girola, et al., Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/ 10.1016/j.bbrc.2015.10.041
N. Girola et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
in the cytosolic levels of Ca2þ, particularly in combination with thapsigargin (Fig. 2B). Finally, we observed that camphene (70 mg/ mL) induced early functional alterations in mitochondria, by partially decreasing the mitochondrial transmembrane potential of tumor cells after 10 min of incubation, followed by loss of the transmembrane potential within 30 min of incubation. In comparison, the positive control CCCP, induced the complete loss of mitochondrial transmembrane potential in tumor cells, after 10 min of incubation with B16F10-Nex2 cells (Fig. 2C).
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3.7. Camphene displays antitumor activity against primary subcutaneously grafted melanoma The antitumor activity of camphene in vivo was evaluated in C57Bl/6 mice previously challenged with 5 104 B16F10-Nex2 cells. Daily doses of camphene (10 mg/mL) in 100 mL PBS, were injected peritumorally. Treatment resulted in a significant decrease in tumor volume as seen on the 16th day extending to the 20th day of treatment with camphene, as compared to the vehicle control (Fig. 4). No toxic effects, loss of weight, or alterations in the animal behavior were observed during camphene treatment.
3.6. Camphene elicits ER-stress proteins and caspase-3 4. Discussion Tumor cells were incubated with camphene at 35 and 70 mg/mL for 3 h and total proteins were extracted as described in methods for evaluation of ER-stress markers by Western blotting. We observed that camphene at 70 mg/mL increased the expression of calreticulin and HmgB1. Simultaneously, camphene significantly increased the expression of caspase-3, a major pro-apoptotic effector enzyme (Fig. 3).
Essential oils provide active components exhibiting antimicrobial, antioxidant and anticancer activity [18]. Previously, we reported that a-pinene induced apoptosis in cancer cells and studied its mechanism of action on murine B16F10-Nex2 melanoma cells and the protective activity against metastatic melanoma [8]. Another monoterpene that showed promising antitumor effects
Fig. 2. ER and mitochondrial disturbances in camphene-treated cells. (a) Camphene induces ER condensation in B16F10-Nex2 cells treated with 70 mg/mL of the monoterpene and stained with fluorescent probe ER-Tracker (Life-technologies™) in red. White arrows indicate high condensation cells (scale bar, 100 mm); (b) Camphene (70 mg/mL) induces ER Ca2þ release in B16F10-Nex2 cells during 40 min of incubation; Ca2þ fluorescence readings were obtained at 10 s intervals; *p < 0.05, for camphene þ T compared to camphene (with no thapsigargin), **p < 0.05, for camphene compared to negative control (without thapsigargin and camphene). (c) Camphene induces collapse of the mitochondrial transmembrane potential (Djm) in B16F10-Nex2 cells during treatment at 70 mg/mL for 10 and 30 min *p < 0.05, compared to negative control. CCCP was used as a positive control.
Please cite this article in press as: N. Girola, et al., Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/ 10.1016/j.bbrc.2015.10.041
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N. Girola et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
Fig. 3. Camphene treatment of B16F10-Nex2 cells enhanced calreticulin, HmgB1 and cleaved caspase-3 levels. Total protein extracts from B16F10-Nex2 previously exposed to 0 (control), 35 or 70 mg/mL camphene were subjected to Western blotting and probed with the respective antibodies. Protein levels were normalized to the actin level.
phosphatidylserine to the outer leaflet of the plasma membrane [22] was also observed in camphene treated melanoma cells. Lipophilic essential oils seem to lack specific cellular targets [23], as they generally pass through the cytoplasmic membrane and render cytotoxic effects, including damage to vital organelles in eukaryotic cells [24], such as mitochondria and the endoplasmic reticulum. Monoterpenes are the main compounds found in the essential oils from plant sources [25], and different biological activities have been attributed to cyclic forms. The amphipathic thymol, for instance, affects cell membrane structures by generating asymmetries and membrane tensions [26]. The apoptotic effects of camphene seem to involve the intrinsic pathway, as confirmed by the loss of mitochondrial membrane potential (Djm) together with activation of caspase-3. The monoterpene induced also significant condensation of ER, an evolutionarily conserved cell stress response [27,28] that can trigger cell death [29]. Camphene induced the increased cytoplasmic release of Ca2þ [30] mainly derived from the ER, as shown by co-incubation with thapsigargin (T), a non-competitive inhibitor of the sarco/ER Ca 2þ ATPase (SERCA) [31]. In addition, the ER stress was also reflected by increased surface expression of HmgB1 and calreticulin [32,33]. Most importantly, camphene was active against subcutaneously grafted B16F10-Nex2 melanoma cells in vivo in a syngeneic model, upon peritumoral administration. Previous reports have already shown that cyclic monoterpenes have great percutaneous absorption [16], enhancing their therapeutic application. We suggest that camphene is a promising cancer therapeutic agent inducing tumor cell apoptosis, as shown in a melanoma model. The potential use of oil components in cancers of difficult surgical access for complete excision has already been discussed in a previous work [12]. Moreover, the increased expression of HmgB1 and calreticulin in apoptotic tumor cells treated with camphene may elicit immunogenic cell death, a prominent pathway for the activation of the immune system [34]. Acknowledgments ~o de Amparo a The present work was supported by Fundaça Pesquisa do Estado de S~ ao Paulo (FAPESP), grant numbers 2015/ 11936-2 and 2011/51739-0, and the Conselho Nacional de Desen gico (CNPq) grant number 300546/ volvimento Científico e Tecnolo 2012-2. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.10.041.
Fig. 4. In vivo antitumor activity of camphene. Six-week-old male C57BL/6 mice were injected subcutaneously with 5 104 B16F10-Nex2 tumor cells. Peritumoral treatment started after the tumor reached 100 mm3. Five animals per group were used. Camphene (10 mg/mL) was injected in 100 mL at 1% DMSO in PBS. Daily doses were given during all treatment period. Tumor size was measured in alternate days with a caliper until the tumor volume reached a maximum of 3000 mm3 when the animals were sacrificed. The control group was treated with vehicle 1% DMSO in PBS. *p < 0.05, indicates significant difference between groups.
was Limonene, studied in preclinical model systems [19]. Presently we focused on camphene and its cytotoxicity against malignant melanoma. Hallmarks of apoptosis were recognized in melanoma cells treated with camphene. They included chromatin condensation, cell shrinkage, formation of apoptotic bodies, nuclear fragmentation, and caspase-3 activation [20,21]. Translocation of
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Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo* Natalia Girola a, *, Carlos R. Figueiredo a, Camyla F. Farias a, Ricardo A. Azevedo b, Adilson K. Ferreira b, Sarah F. Teixeira b, Tabata M. Capello c, Euder G.A. Martins d, ~o H.G. Lago c Alisson L. Matsuo e, Luiz R. Travassos a, Joa ~o Paulo, SP, Brazil Experimental Oncology Unit (UNONEX), Federal University of Sa ~o Paulo, SP, Brazil Laboratory of Tumor Immunology, Institute of Biomedical Sciences, University of Sa c ~o Paulo, SP, Brazil Institute of Environmental, Chemical and Pharmaceutical Sciences, Federal University of Sa d ~o Paulo, SP, Brazil Institute of Biosciences, University of Sa e ~o Paulo, SP, Brazil Interdepartmental Group of Health Economics (Grides), Federal University of Sa a
b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 3 October 2015 Accepted 8 October 2015 Available online xxx
Natural monoterpenes were isolated from the essential oil of Piper cernuum Vell. (Piperaceae) leaves. The crude oil and the individual monoterpenes were tested for cytotoxicity in human tumor cell lineages and B16F10-Nex2 murine melanoma cells. In the present work we demonstrate the activity of camphene against different cancer cells, with its mechanism of action being investigated in vitro and in vivo in murine melanoma. Camphene induced apoptosis by the intrinsic pathway in melanoma cells mainly by causing endoplasmic reticulum (ER) stress, with release of Ca2þ together with HmgB1 and calreticulin, loss of mitochondrial membrane potential and up regulation of caspase-3 activity. Importantly, camphene exerted antitumor activity in vivo by inhibiting subcutaneous tumor growth of highly aggressive melanoma cells in a syngeneic model, suggesting a promising role of this compound in cancer therapy. © 2015 Elsevier Inc. All rights reserved.
Keywords: Camphene Melanoma Apoptosis Antitumor
1. Introduction Malignant melanoma is a most aggressive skin cancer and in its metastatic form, is highly resistant to conventional therapeutic agents. Immunomodulatory antibodies are promising therapeutic agents but may raise adverse collateral effects, therefore, new molecules from natural sources are constantly being studied, representing alternatives for drug development [1e4]. Common anticancer effects of natural products include inhibition of cell proliferation and of signaling pathways, induction of apoptosis, de-
*
Camphene induces intrinsic apoptosis on melanoma cells. * Corresponding author. Unidade de Oncologia Experimental, Departamento de ~o Paulo, Rua Microbiologia, Imunologia e Parasitologia, Universidade Federal de Sa ~o Paulo, SP 04023-062, Brazil. Botucatu 862, 8 andar, Sa E-mail address: [email protected] (N. Girola).
stabilization of cytoskeleton integrity, cell cycle modulation, cell migration and metastasis inhibition. These compounds are thus important tools in chemical and pharmacological investigation [3,5,6]. Presently, we characterized the in vitro cytotoxic activity on different tumor cells of the crude essential oil extracted from leaves of Piper cernuum and its monoterpene constituents. Anticancer activities have been attributed to monoterpenes [7], and a-pinene and limonene have been well characterized [8e10], but the antitumor activity of camphene has not been reported before. In this work we observed that camphene induced intrinsic apoptosis in B16F10-Nex2 melanoma cells following the endoplasmic reticulum (ER) stress with liberation of calcium, chromatin and chaperon proteins, as well as caspase-3 activation. A promising antitumor activity was seen in vivo, using a syngeneic melanoma model. All these results suggest that camphene is a candidate for cancertherapy drug development.
http://dx.doi.org/10.1016/j.bbrc.2015.10.041 0006-291X/© 2015 Elsevier Inc. All rights reserved.
Please cite this article in press as: N. Girola, et al., Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/ 10.1016/j.bbrc.2015.10.041
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N. Girola et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
2. Materials and methods 2.1. Plant material and extraction procedures
values in comparison to untreated cells as previously described [13]. Cisplatin was used for comparison of positive toxicity. All experiments were performed in triplicates.
gico do Branches of P. cernuum were collected at Parque Ecolo ~o, Sa ~o Paulo State, Brazil (Latitude: 23 530 4200 Pereque, Cubata South and Longitude: 46 250 3000 West) in July, 2012. For extraction of crude essential oil, fresh branches (200 g) were subjected to steam distillation in a Clevenger type apparatus for 4 h, giving 320 mg of a pale yellow essential oil (yield 0.16%). The oil was dried at room temperature and stored in a sealed vial under refrigeration prior to analysis.
B16F10-Nex2 cells (5 105 cells/well) were cultured on 6-well plates and incubated with camphene at 70 mg/mL at 37 C for 3 h, 4 h and 5 h. DNA was extracted using the TELT buffer (50 mM TriseHCl pH 8.0, Triton X-100 0.4%, 2.5 mM EDTA pH 9.0, and 2.5 M LiCl) and analyzed in Agarose gel electrophoresis as previously described [14].
2.2. Isolation of main compounds from essential oil
2.6. Chromatin condensation analysis
Part of the crude essential oil from branches of P. cernuum (300 mg) was subjected to CC on silica gel eluted with CH2Cl2 and CH2Cl2:MeOH (98:2, 95:5 and 9:1) to afford 67 fractions (6 mL each), which were individually analyzed by FID-GC. These fractions were pooled in ten groups (A1 e A10). Groups A3 (12 mg) and A4 (7 mg) were individually purified by prep. TLC (SiO2 e CH2Cl2: MeOH 99:1) to afford carvacrol (8 mg) and tymol (3 mg), respectively. Group A6 (8 mg) was composed by pure a-terpineol while group A8 (10 mg) was purified by prep. TLC (SiO2 e CH2Cl2: MeOH 98:2) to afford linalol (6 mg). Groups A1 (75 mg) and A2 (52 mg) were pooled and subjected to CC on silica gel soaked with AgNO3 (15%) eluted with CH2Cl2 and CH2Cl2: acetone (99:1 and 98:2) to afford 70 fractions (6 mL each), which were individually analyzed by FID-GC. These fractions were pooled in seven groups (B1 e B7). Groups B3 (3 mg) and B4 (32 mg) were composed by pure a-pinene and camphene. Prep. TLC (SiO2/AgNO3 15% e CH2Cl2:acetone 99:1) separation of group B5 afforded limonene (2 mg) and myrcene (3 mg) while group B7 was composed by pure p-cymene (4 mg). All isolated compounds displayed purity higher than 98% (FID-CG analysis). The identification of the components was based on the comparison of their mass spectra with those in the spectrometer data base (Willey 229 library) and confirmed by determination of their Kovats indexes, which were determined relative to the retention times of a series of n-alkanes [11]. The oils and isolated compounds were stored in DMSO at e 20 C (DMSO, SIGMA).
B16F10-Nex2 cells (1 104) were cultivated overnight on Hi-Q4 dish and incubated with camphene at 70 mg/mL for 5 h. After incubation, cells were stained with 1.25 mM Hoechst 33342 (Invitrogen, Carlsbad, CA) for 10 min, washed two times with PBS 1X and further examined by fluorescence microscopy in a Nikon BioStation IM microscope. Images were processed with ImageJ software (V 1.46r, National Institutes of Health, USA).
2.3. Cell lines and culture conditions The murine melanoma cell line B16F10-Nex2, is a melanotic, aggressive sub-line derived from an original B16F10 melanoma cell line provided by the Ludwig Institute for Cancer Research (LICR), ~o Paulo branch, and was established at the Experimental Sa ~o Paulo (UNIFESP), and Oncology Unit, Federal University of Sa lulas do Rio de Janeiro (BCRJ), reg. deposited in the Banco de Ce 0342. The following human cancer cell lines: melanoma A2058, breast cancer SKBR-3, cervical cancer HeLa, human myeloid leukemia HL-60, were provided by the LICR. The human glioblastoma U87-MG cell line was provided by Prof. O. Keith Okamoto, from ~o Paulo. Cells were cultivated as previously University of Sa described [12]. 2.4. Cell viability assay Crude essential oil from branches of P. cernuum and isolated monoterpenes were incubated with 1 104 tumor and nontumorigenic cells for 24 h at different concentrations ranging from 0 to 100 mg/mL. Cell viability was quantified using MTT (3[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) (SigmaeAldrich, St. Louis, MO, USA), and was quantified as percent
2.5. DNA degradation
2.7. Mitochondrial transmembrane potential (Djm) B16F10-Nex2 cells (1 105) were incubated with 5 nM of tetramethylrhodamine ethyl ester (TMRE, Molecular Probes, OR, USA), a fluorescent probe for intact mitochondrial transmembrane potential, for 30 min at 37 C with 5% CO2 in different 5 mL FACs tubes. Subsequently, 70 mg/mL of camphene, 100 mM of the positive carbonyl cyanide 3-chlorophenylhydrazone (CCCP, SigmaeAldrich, St. Louis, MO, USA), and negative control (RPMI medium) were added into separated cell tubes and incubated at 37 C. CCCP is a well-known protonophore used for positive mitochondrial transmembrane potential disruption [15]. Fluorescence readings were performed after 10 and 30 min of incubation in a FACSCalibur (BD Bioscience, Franklin Lakes, NJ) and further analyzed using the FlowJo software (TreeStar Inc., Ashland, OR, USA). 2.8. Calcium release Release of Ca2þ from ER, upon treatment with camphene was detected using the Fluo 4 NW kit (Invitrogen). B16F10-Nex2 (5 104) cells were seeded in 96-well plates and processed according to the manufacturer's instructions. Then, cells were incubated with or without camphene at 70 mg/mL in the presence or absence of 2 mM of thapsigargin (T) (Enzo Life Science) and fluorescence values were obtained every 60 s, for 1 h, in a plate spectrophotometer (Spectra Max M2e, Molecular Devices). 2.9. Endoplasmic reticulum stress B16F10-Nex2 cells (2 104) were incubated with camphene in four chamber dishes at 70 mg/mL and the ER integrity was assessed by the ER red fluorescent probe ER-Tracker (Life-technologiesTM) following the manufacturer's instructions. After incubation, tumor cells were incubated with ER-Tracker for 10 min at 37 C, and then tumor cells were washed in PBS and immediately observed in a fluorescence microscope (BioStation, Nikon). 2.10. Annexin V assay B16F10-Nex2 cells (5 105) were cultured in 6-well plates and further incubated with camphene 70 mg/mL or complete medium (negative control) for 5 h at 37 C. Phosphatidylserine detection
Please cite this article in press as: N. Girola, et al., Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/ 10.1016/j.bbrc.2015.10.041
N. Girola et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
was performed using the Annexin V-FITC Apoptosis Detection Kit (SigmaeAldrich, St. Louis, MO) as previously described [16]. Quantification of live cells (AV/PI), necrotic cells (AV/PIþ), early apoptotic cells (AVþ/PI) and late apoptotic cells (AVþ/PIþ), was performed using FlowJo software (version 9.5.3, Tree Star Inc., Ashland, OR, USA). 2.11. In vivo subcutaneous melanoma growth Male C57Bl/6 mice were subcutaneously grafted in the flank with 5 104 B16F10-Nex2 cells. After 12 days of tumor challenge, animals were treated with 10 daily peritumoral doses of 300 mg (total 10 mg/kg) of camphene. DMSO (1%) in PBS was used as vehicle control. Mice were euthanized when the maximum allowed volume of 3000 mm3 was achieved. Tumor volume was calculated and analyzed as previously described [12]. Animal experiments were carried out according with the UNIFESP Ethics Committee for Animal Experimentation. 2.12. Cell protein extraction and Western blotting B16F10-Nex2 cells (106) were treated with 35 or 70 mg/mL of camphene for 3 h. After treatment, proteins in cell lysates were analyzed by Western blotting as described elsewhere [17]. The following antibodies were used: anti-b-actin, anti-HmgB1, anticaspase-3 or cleaved caspase-3 and anti-calreticulin, all purchased from Cell Signaling Technology (Beverly, MA, USA). b-Actin was used as loading control. Secondary antibodies conjugated with IgG horseradish peroxidase were purchased from SigmaeAldrich (St. Louis, MO, USA).
3
Table 1 IC50 values obtained for crude essential oil from branches of Piper cernuum against different tumor cells in vitro. Lineages
IC50 ± S.D. (mg/mL) Crude essential oil
B16F10-Nex2 A2058 HeLa HL-60 U87-MG
39 24.6 23.6 15.5 19.0
± ± ± ± ±
cisplatin
2.0 1.1 0.1 3.5 0.0
53 43 20.4 21.2 45.0
± ± ± ± ±
4.0 1.5 1.2 1.5 6.0
3.3. Cytotoxic effects of camphene in different tumor and nontumorigenic cell lines The cytotoxic effects of camphene (IC50) were evaluated in tumor and non-tumorigenic cell lines as shown in Table 3. The concentration of 70 mg/mL was therefore used in the subsequent experiments with B16F10-Nex2 melanoma cells. 3.4. Camphene induces apoptotic hallmarks in murine melanoma B16F10-Nex2 cells At 70 mg/mL, camphene induced cell shrinkage and DNA Table 2 IC50 values obtained in B16F10-Nex2 cell for different compounds from essential oil from branches of Piper cernuum and its chemical structures of monoterpenes. Monoterpene
Structural formula
IC50 ± S.D. (mg/mL)
a-pinene
24 ± 0.4
Camphene
70 ± 4.0
Limonene
59 ± 3.0
Carvacrol
82 ± 4.0
Tymol
88 ± 0.7
Myrcene
>100
p-cymene
>100
a-terpineol
>100
Linalol
>100
2.13. Statistical analysis The experiments were performed in triplicate and the values are expressed as means ± standard deviations (S.D.). Student's t test was used for statistical analysis of in vitro and in vivo experiments. For significance analyses GraphPad Prism 4.0 software was used (La Jolla, CA); p < 0.05 was considered a significant difference. 3. Results 3.1. Cytotoxicity of the essential oil from branches of P. cernuum The cytotoxicity of the essential oil from branches of P. cernnum was evaluated in murine and human cancer cells, as shown in Table 1. The IC50 values were obtained and compared with those for cisplatin, taken as a positive control, against the same cells. The crude essential oil was most cytotoxic in HL-60 and U87-MG cell lines, and with the exception of Hela cells was generally more active than cisplatin. 3.2. Natural monoterpenes from P. cernuum and their cytotoxicity against different tumor cells Monoterpenes isolated from P. cernuum were a-pinene, camphene, limonene, carvacrol, tymol, myrcene, p-cymene, aterpineol, and linalol. Their structure formulas and cytotoxicity against B16F10-Nex2 melanoma cells are shown in Table 2. apinene and limonene were the most cytotoxic species that have already been tested in biological systems. Camphene comes next, and its anti-tumor activity has been studied for the first time in the present work.
Please cite this article in press as: N. Girola, et al., Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/ 10.1016/j.bbrc.2015.10.041
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N. Girola et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7 Table 3 IC50 ± S.D. values (mg/mL) for camphene in different cancer cell lines. Lineages
Camphene
B16F10-Nex2 A2058 HeLa HL-60 U87-MG SKBR-3
71.2 35.2 110.1 27.0 55.4 34.7
± ± ± ± ± ±
4.3 1.2 5.1 2.0 1.6 2.5
condensation in tumor cells, with pyknotic nuclei indicated by yellow arrows in Fig. 1A. DNA fragmentation was also evidenced by Agarose gel electrophoresis (Fig. 1B). Camphene also induced significant exposure of phosphatidylserine after 5 h incubation with
melanoma cells, as shown by positive Annexin-V FITC staining, and denoting early apoptosis (Fig. 1C). At the same time half of the cells double stained with Annexin-V and PI indicating membrane damage or late apoptosis as previously described [8]. 3.5. Endoplasmic reticulum (ER) and mitochondrial alterations induced by camphene Camphene caused disturbances in the endoplasmic reticulum and mitochondria of tumor cells, both related to the intrinsic apoptotic process. We observed that camphene induced significant condensation of ER in B16F10-Nex2 cells (80% of positive cells) after treatment with 70 mg/mL, as compared to the negative control (30% of positive cells) (Fig. 2A). This was shown by high fluorescence of cells incubated with the ER-specific red fluorescence probe, ERtracker. We observed also that camphene promoted an increase
Fig. 1. Cell morphology and DNA degradation in camphene-treated melanoma cells. (a) Evaluation of chromatin condensation in camphene-treated cells. B16F10-Nex2 cells were treated with camphene at 70 mg/ml for 5 h and analyzed by fluorescence microscopy (scale bars, 100 mm). (b) Agarose gel electrophoresis of DNA fragmentation in B16F10-Nex2 cells induced by 70 mg/mL camphene at 37 C at different times (3h, 4h and 5 h). (c) Annexin V and PI labeling. B16F10-Nex2 cells were treated with 70 mg/mL of camphene for 5 h and stained with Annexin V-FITC and PI. Analysis was performed by fluorescence microscopy. The values are percentages obtained from three independent experiments. *p < 0.05, compared to negative control.
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N. Girola et al. / Biochemical and Biophysical Research Communications xxx (2015) 1e7
in the cytosolic levels of Ca2þ, particularly in combination with thapsigargin (Fig. 2B). Finally, we observed that camphene (70 mg/ mL) induced early functional alterations in mitochondria, by partially decreasing the mitochondrial transmembrane potential of tumor cells after 10 min of incubation, followed by loss of the transmembrane potential within 30 min of incubation. In comparison, the positive control CCCP, induced the complete loss of mitochondrial transmembrane potential in tumor cells, after 10 min of incubation with B16F10-Nex2 cells (Fig. 2C).
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3.7. Camphene displays antitumor activity against primary subcutaneously grafted melanoma The antitumor activity of camphene in vivo was evaluated in C57Bl/6 mice previously challenged with 5 104 B16F10-Nex2 cells. Daily doses of camphene (10 mg/mL) in 100 mL PBS, were injected peritumorally. Treatment resulted in a significant decrease in tumor volume as seen on the 16th day extending to the 20th day of treatment with camphene, as compared to the vehicle control (Fig. 4). No toxic effects, loss of weight, or alterations in the animal behavior were observed during camphene treatment.
3.6. Camphene elicits ER-stress proteins and caspase-3 4. Discussion Tumor cells were incubated with camphene at 35 and 70 mg/mL for 3 h and total proteins were extracted as described in methods for evaluation of ER-stress markers by Western blotting. We observed that camphene at 70 mg/mL increased the expression of calreticulin and HmgB1. Simultaneously, camphene significantly increased the expression of caspase-3, a major pro-apoptotic effector enzyme (Fig. 3).
Essential oils provide active components exhibiting antimicrobial, antioxidant and anticancer activity [18]. Previously, we reported that a-pinene induced apoptosis in cancer cells and studied its mechanism of action on murine B16F10-Nex2 melanoma cells and the protective activity against metastatic melanoma [8]. Another monoterpene that showed promising antitumor effects
Fig. 2. ER and mitochondrial disturbances in camphene-treated cells. (a) Camphene induces ER condensation in B16F10-Nex2 cells treated with 70 mg/mL of the monoterpene and stained with fluorescent probe ER-Tracker (Life-technologies™) in red. White arrows indicate high condensation cells (scale bar, 100 mm); (b) Camphene (70 mg/mL) induces ER Ca2þ release in B16F10-Nex2 cells during 40 min of incubation; Ca2þ fluorescence readings were obtained at 10 s intervals; *p < 0.05, for camphene þ T compared to camphene (with no thapsigargin), **p < 0.05, for camphene compared to negative control (without thapsigargin and camphene). (c) Camphene induces collapse of the mitochondrial transmembrane potential (Djm) in B16F10-Nex2 cells during treatment at 70 mg/mL for 10 and 30 min *p < 0.05, compared to negative control. CCCP was used as a positive control.
Please cite this article in press as: N. Girola, et al., Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/ 10.1016/j.bbrc.2015.10.041
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Fig. 3. Camphene treatment of B16F10-Nex2 cells enhanced calreticulin, HmgB1 and cleaved caspase-3 levels. Total protein extracts from B16F10-Nex2 previously exposed to 0 (control), 35 or 70 mg/mL camphene were subjected to Western blotting and probed with the respective antibodies. Protein levels were normalized to the actin level.
phosphatidylserine to the outer leaflet of the plasma membrane [22] was also observed in camphene treated melanoma cells. Lipophilic essential oils seem to lack specific cellular targets [23], as they generally pass through the cytoplasmic membrane and render cytotoxic effects, including damage to vital organelles in eukaryotic cells [24], such as mitochondria and the endoplasmic reticulum. Monoterpenes are the main compounds found in the essential oils from plant sources [25], and different biological activities have been attributed to cyclic forms. The amphipathic thymol, for instance, affects cell membrane structures by generating asymmetries and membrane tensions [26]. The apoptotic effects of camphene seem to involve the intrinsic pathway, as confirmed by the loss of mitochondrial membrane potential (Djm) together with activation of caspase-3. The monoterpene induced also significant condensation of ER, an evolutionarily conserved cell stress response [27,28] that can trigger cell death [29]. Camphene induced the increased cytoplasmic release of Ca2þ [30] mainly derived from the ER, as shown by co-incubation with thapsigargin (T), a non-competitive inhibitor of the sarco/ER Ca 2þ ATPase (SERCA) [31]. In addition, the ER stress was also reflected by increased surface expression of HmgB1 and calreticulin [32,33]. Most importantly, camphene was active against subcutaneously grafted B16F10-Nex2 melanoma cells in vivo in a syngeneic model, upon peritumoral administration. Previous reports have already shown that cyclic monoterpenes have great percutaneous absorption [16], enhancing their therapeutic application. We suggest that camphene is a promising cancer therapeutic agent inducing tumor cell apoptosis, as shown in a melanoma model. The potential use of oil components in cancers of difficult surgical access for complete excision has already been discussed in a previous work [12]. Moreover, the increased expression of HmgB1 and calreticulin in apoptotic tumor cells treated with camphene may elicit immunogenic cell death, a prominent pathway for the activation of the immune system [34]. Acknowledgments ~o de Amparo a The present work was supported by Fundaça Pesquisa do Estado de S~ ao Paulo (FAPESP), grant numbers 2015/ 11936-2 and 2011/51739-0, and the Conselho Nacional de Desen gico (CNPq) grant number 300546/ volvimento Científico e Tecnolo 2012-2. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.10.041.
Fig. 4. In vivo antitumor activity of camphene. Six-week-old male C57BL/6 mice were injected subcutaneously with 5 104 B16F10-Nex2 tumor cells. Peritumoral treatment started after the tumor reached 100 mm3. Five animals per group were used. Camphene (10 mg/mL) was injected in 100 mL at 1% DMSO in PBS. Daily doses were given during all treatment period. Tumor size was measured in alternate days with a caliper until the tumor volume reached a maximum of 3000 mm3 when the animals were sacrificed. The control group was treated with vehicle 1% DMSO in PBS. *p < 0.05, indicates significant difference between groups.
was Limonene, studied in preclinical model systems [19]. Presently we focused on camphene and its cytotoxicity against malignant melanoma. Hallmarks of apoptosis were recognized in melanoma cells treated with camphene. They included chromatin condensation, cell shrinkage, formation of apoptotic bodies, nuclear fragmentation, and caspase-3 activation [20,21]. Translocation of
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Please cite this article in press as: N. Girola, et al., Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/ 10.1016/j.bbrc.2015.10.041
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Please cite this article in press as: N. Girola, et al., Camphene isolated from essential oil of Piper cernuum (Piperaceae) induces intrinsic apoptosis in melanoma cells and displays antitumor activity in vivo, Biochemical and Biophysical Research Communications (2015), http://dx.doi.org/ 10.1016/j.bbrc.2015.10.041