The redox antimalarial dihydroartemisinin targets human metastatic melanoma cells but not primary melanocytes with induction of NOXA-dependent apoptosis

Chemicals

All chemicals were purchased from Sigma Chemical Co (St. Louis, MO, USA).

Cell culture

G-361, A375, and LOX human melanoma cells and dermal neonatal foreskin Hs27 fibroblasts from ATCC (Manassas, VA, USA) were cultured as published recently [19–21]. Primary human epidermal melanocytes (adult skin, lightly pigmented: HEMa-LP from Cascade Biologics, abbreviated HEMa) were cultured using Medium 154 medium supplemented with HMGS-2 growth supplement. Cells were maintained at 37°C in 5% CO₂, 95% air in a humidified incubator.

Transmission electron microscopy

Cells were fixed in situ with 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH7.4), postfixed in 1% osmium tetroxide in cacodylate buffer and then processed and analyzed as published recently [22].

Human stress and toxicity pathfinder™ RT² profiler™ PCR expression array

After pharmacological exposure, total cellular RNA (3×10⁶ A375 cells) was prepared according to a standard procedure using the RNeasy kit (Qiagen, Valencia, CA, USA). Reverse transcription was performed using the RT² First Strand kit (Superarray, Frederick, MD, USA) and 5 μg total RNA as described previously [20]. The RT² Human Stress and Toxicity Pathfinder^™ PCR Expression Array (SuperArray) profiling the expression of 84 stress-related genes was run and analyzed as published earlier [20, 21, 23]. Expression values were averaged across three independent array experiments, and standard deviation was calculated for graphing.

CDKN1A, DDIT3, GADD45A, HMOX1, and PMAIP1 expression analysis by real time RT-PCR

For expression analysis by real time RT-PCR, total cellular RNA (3×10⁶ cells) was prepared using the RNEasy kit from Qiagen (Valencia, CA, USA) and processed according to published standard procedures [21, 23]. After reverse transcription, real time RT-PCR was performed using an Applied Biosystems 7000 SDS and Applied Biosystems’ Assays On Demand primers specific to CDKN1A (assay ID Hs00355782_m1), DDIT3 (assay ID Hs00358796_g1), GADD45A (assay ID Hs00169255_m1), HMOX1 (assay ID Hs00157965_m1), PMAIP1 (assay ID Hs00560402_m1), and GAPDH (assay ID Hs99999905_m1). Gene-specific product was normalized to GAPDH and quantified using the comparative (ΔΔC_t) Ct method as described in the ABI Prism 7000 sequence detection system user guide [23]. Expression values were averaged across three independent experiments, and standard deviation was calculated for graphing.

siRNA-Transfection targeting PMAIP1 expression

A375 cells were transiently transfected with a 100 nmol pool of four small interfering RNA (siRNA) oligonucleotides (oligos) targeting PMAIP1 or a 100 nmol pool of four nontargeting siRNA oligos using the DharmaFECT 1 transfection reagent (Dharmacon RNA Technologies, Lafayette, Colorado, USA) following a standard procedure [20]. The sequences of siGENOME PMAIP1 SMARTpool (PMAIP1 siRNA) (Gen-Bank: NM 021127) were AAACUGAACUUCCGGCAGA, AUUCUGUAUCCAAACUCU, CUGGAAGUCGAGU GUGCUA, and GCAAGAACGCUCAACCGAG.

Immunoblot analysis of NOXA, PUMA, HO-1, CHOP, and p21

Sample preparation, SDS-PAGE, transfer to nitrocellulose, and development occurred as described earlier [20, 21]. Gel percentages were 12% (HO-1, p21, CHOP) and 15% (NOXA, PUMA). The following primary antibodies were used: mouse anti-CHOP monoclonal antibody (2895S; 1:1,000; Cell Signaling Technology, Danvers, MA); rabbit anti-HO-1 polyclonal antibody (SPA-896F, 1:5,000; Stress-gen Bioreagents, Ann Arbor, MI); monoclonal mouse anti-NOXA IgG (OP180; 1:1,000; EMD Chemicals, Gibbstown, NJ); polyclonal rabbit anti-PUMA antibody (4976; 1:1,000; Cell Signaling Technology) and mouse anti-p21 monoclonal antibody (2946; 1:2,000; Cell Signaling Technologies).

Cell death analysis

Viability and induction of cell death (early and late apoptosis/necrosis) were examined by annexin-V-FITC (AV)/propidium iodide (PI) dual staining of cells followed by flow cytometric analysis as published previously [19].

Flow cytometric detection of cleaved procaspase-3, phospho-p53 (Ser15), and phospho-H2A.X

Treatment-induced proteolytic caspase-3 activation and formation of phospho-p53 (Ser15) and phospho-H2A.X were examined in cultured A375 human melanoma cells using antibodies directed against cleaved/activated caspase-3 (Asp 175), phospho-p53 (Ser15), and phospho-histone H2A.X (Ser139) (Alexa Fluor 488 conjugates, Cell Signaling, Danvers, MA, USA) followed by flow cytometric analysis as published recently [19, 23].

Detection of intracellular oxidative stress by flow cytometric analysis

Induction of intracellular oxidative stress by DHA was analyzed by flow cytometry using 2′,7′-dichlorodihy-drofluorescein diacetate (DCFH-DA) as a sensitive non-fluorescent precursor dye according to a published standard procedure [19].

Determination of reduced cellular glutathione content

Intracellular reduced glutathione was measured using the GSH-Glo Glutathione assay kit (Promega; San Luis Obispo, CA) as described recently [23]. Data are normalized to GSH content in untreated cells and expressed as means ± SD (n=3).

Mitochondrial transmembrane potential

Mitochondrial transmembrane potential (Δψm) was assessed using the potentiometric dye 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethyl-benzimidazolyl-carbocyanine iodide (JC-1) following our published flow cytometric procedure [19].

Cell Glo ATP assay

ATP content per 10,000 cells was determined using the CellTiter-Glo luminescent assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions as published earlier [23]. Data are normalized to ATP content in untreated cells (expressed as means ± SD (n=3).

Comet assay (alkaline single cell electrophoresis)

The alkaline comet assay was performed on A375 melanoma and HEMa cells according to the manufacturer’s instructions (Trevigen, Gaithersburg, MD, USA) as published in detail recently [23]. Cells were stained with SYBR^™ Green and then visualized and analyzed using a fluorescence microscope (fluorescein filter) and CASP software. At least 75 tail moments for each group were analyzed in order to calculate the mean ± S.D. for each group.

Statistical analysis

Unless indicated differently, the results are presented as mean ± S.D. of at least three independent experiments. All data were analyzed employing one-way analysis of variance (ANOVA) with Tukey’s post hoc test using the Prism 4.0 software. Differences were considered significant at p≤0.05. Means without a common letter differ (p<0.05).

DHA induces cell death in human A375 metastatic melanoma cells but not in primary epidermal melanocytes

First, induction of cell death by DHA was assessed by flow cytometric analysis of annexinV-FITC/propidium iodide-stained metastatic melanoma cell lines (A375, G361, LOX), primary human fibroblasts (Hs27), and primary epidermal melanocytes (HEMa) (Fig. 1). Exposure to DHA (10 and 40 μM, 48 h) dose-dependently induced pronounced cell death in metastatic melanoma cells (Fig. 1a–d), but no impairment of viability was observed in primary epidermal melanocytes (Fig. 1a and d) and fibroblasts (Fig. 1c and d) exposed to DHA concentrations up to 100 μM.

Array analysis reveals upregulation of oxidative and genotoxic stress response gene expression in A375 melanoma cells exposed to DHA

Next, modulation of stress and toxicity response gene expression was examined in A375 cells exposed to DHA (40 μM, 48 h exposure) using the RT² Human Stress and Toxicity Profiler^™ PCR Expression Array technology (SuperArray, Frederick, MD) (Fig. 2a). Out of 84 stress-related genes contained on the array DHA-induced expression changes in A375 cells affected 21 genes by at least two-fold over untreated control cells as summarized in Fig. 2b. More than fivefold upregulation was detected affecting the p53-regulated DNA damage inducible genes growth arrest and DNA-damage-inducible, alpha (GADD45A; 6-fold), cyclin-dependent kinase inhibitor 1 (CDKN1A; 6-fold), and DNA damage inducible transcript 3 (DDIT3/GADD153; 8-fold). Moreover, pronounced upregulation of genes encoding the antioxidant enzyme heme oxygenase-1 (encoded by HMOX1; 6-fold), the oxidative stress-responsive transcription factor and tumor suppressor early growth response protein 1 (encoded by EGR1; 5-fold), and the cytokine granulocte-macrophage colony stimulating factor 2 (CSF2; 6-fold) was observed.

DHA induces oxidative stress in A375 melanoma cells

Led by upregulated expression of oxidative stress response genes after 48 h continuous exposure (HMOX1, EGR1; Fig. 2), we further examined the occurrence of cellular oxidative stress and redox alterations in DHA-treated A375 melanoma cells (Fig. 3).

Time course analysis of HMOX1 expression was performed by quantitative RT-PCR and detection revealing pronounced upregulation within 12 h continuous exposure (Fig. 3a). In addition, in A375 cells exposed to DHA (20 and 40 μM) significant depletion of intracellular reduced glutathione levels was observed at a similar time point (12 h; Fig. 3b). A dose-dependent elevation of intracellular oxidative stress could be observed in A375 cells exposed to DHA (10–40 μM, 24 h) as assessed by 2′,7′-dichloro-dihydrofluorescein diacetate detection of intracellular peroxide levels using flow cytometry (Fig. 3c). Over a 24 h treatment period (DHA, 40 μM), DCF fluorescence intensity increased approximately fivefold and was significantly upregulated within 12 h continuous exposure (Fig. 3d). Similar results were observed in other melanoma cell lines (G361; Fig. 3d). In contrast, DCF fluorescence intensity remained unaltered in primary melanocytes (HEMa) and Hs27 fibroblasts exposed to DHA (40 μM; 24 h; Fig. 3d), suggesting that DHA treatment does not induce oxidative stress in cells that are resistant to DHA-induced cell death (Fig. 1d). Further DCF-based analysis revealed that DHA-induced intracellular oxidative stress was blocked if A375 cells were pretreated with the thiol-antioxidant N-acetyl-L-cysteine (NAC, 10 mM, 24 h pretreatment followed by 40 μM DHA, 24 h) or the iron chelator deferoxamine (DFO, 20 μM, 1 h pretreatment followed by 40 μM DHA, 24 h) (Fig. 3e). In contrast, treatment with the pan-caspase inhibitor zVADfmk that was able to protect A375 cells against DHA-induced cell death (as detailed further in Fig. 4b) did not suppress elevation of cellular DCF fluorescence suggesting that DHA-induction of intracellular oxidative stress is an early event that does not occur downstream of caspase activation and cellular disintegration.

DHA-induced A375 melanoma cell apoptosis with mitochondrial impairment and caspase 3 activation is antagonized by iron chelation and antioxidant intervention

An apoptotic mode of DHA-induced A375 cell death was suggested by flow cytometric detection of annexin V-positivity, a cellular marker indicative of early stages of apoptosis that involve phosphatidylserine externalization at the plasma membrane level (Fig. 1a and b and Fig. 4b and d). Further morphological evidence in support of DHA-induced apoptosis was obtained employing transmission electron microsocopy that revealed early membrane blebbing (24 h exposure) followed by nuclear condensation (48 h exposure), both established hallmarks of cellular apoptosis (Fig. 4a) [24]. Consistent with caspase-dependent execution of DHA-induced cell death, cell viability was maintained upon cotreatment with the pancaspase inhibitor zVADfmk, an effect also observed with G361 melanoma cells (Fig. 4b).

Further flow cytometric analysis using a cleaved-procaspase 3-directed Alexa488-conjugated antibody revealed proteolytic activation of this executioner caspase that occurred dose dependently (10–40 μM DHA, 24 h; Fig. 4c). Consistent with a causative involvement of cellular oxidative stress in DHA-induced melanoma cell apoptosis, antioxidant pretreatment (NAC, 10 mM, 24 h preincubation) significantly diminished cytotoxicity of DHA (Fig. 4b), and significant sensitization towards DHA-induced cytotoxicity (20 μM, 24 h exposure) was observed in A375 cells that were pre-exposed to the prooxidant inhibitor of glutathione biosynthesis L-buthionine-S,R-sulfoximine (BSO, 1 mM; 24 h preincubation; Fig. 4d) [20]. Furthermore, iron chelation (DFO, 20 μM, 1 h pretreatment) efficiently suppressed DHA-induced cytotoxicity in A375 and G361 melanoma cells (Fig. 4b), and DHA-dependent proteolytic caspase 3 activation was completely blocked by DFO in A375 cells (Fig. 4c).

Further flow cytometric analysis using the sensor dye JC-1 revealed an early impairment of mitochondrial integrity as obvious from dramatic loss of transmembrane potential (Δψm) that occurred within 12 h in DHA exposed A375 cells (Fig. 4f). In the context of mitochondrial impairment, DHA-induced energy crisis was evident from cellular ATP depletion in A375 cells that reached the level of statistical sigificance within 12 exposure time (40 μM DHA; Fig. 4e). None of these DHA-induced changes observed in A375 cells (AV-PI positivity, EM morphological changes, procaspase 3 cleavage, loss of Δψ_m and ATP) was observed in melanocytes or Hs27 fibroblasts that maintained full viability during the course of the experiment (up to 48 h continuous exposure to DHA, 40 μM, Fig. 1a and d, and data not shown).

DHA-induced early upregulation of PMAIP1 (NOXA) expression occurs in A375 and G361 melanoma cells but not in primary melanocytes

Our expression array data indicated that DHA exposure occurred with transcriptional upregulation of PMAIP1 encoding the proapoptotic BH3-only regulator protein NOXA (Fig. 2). We therefore examined DHA-induced PMAIP1 expression at the mRNA and protein level as a function of exposure time in A375 and G361 melanoma cells versus primary melanocytes (Fig. 5a–f). Time course analysis revealed significant upregulation of PMAIP1 transcript levels that occurred in both melanoma cell lines within 6 h continuous exposure (DHA, 40 μM) but was not observed in melanocytes (Fig. 5a–c). Immunoblot detection of NOXA confirmed DHA-induction of PMAIP1 expression at the protein level in A375 and G361 cells (Fig. 5d–f). In contrast, NOXA protein levels remained undetectable in melanocytes (Fig. 5f, left panel). In parallel with dramatic upregulation of NOXA protein levels as an early response to DHA exposure observed in melanoma cells, immunoblot detection in A375 cells also revealed a moderate upregulation of the proapoptotic BH3-only mediator PUMA that remained unchanged in DHA-treated melanocytes (Fig. 5d and f).

Genetic antagonism of PMAIP1 (NOXA) expression protects A375 metastatic melanoma cells from DHA-induced apoptosis

Based on the established key role of NOXA in the mitochondrial pathway of apoptosis [25–27], we then tested the hypothesis that upregulation of PMAIP1 expression may be causatively involved in DHA-induced A375 melanoma cell apoptosis. To this end, genetic target modulation using a siRNA approach targeting PMAIP1 expression was employed. First, efficacy of PMAIP1 knockdown (siPMAIP1) was confirmed at the protein level (Fig. 5g). Immunoblot detection revealed that DHA-induced upregulation of NOXA protein levels, as observed earlier in wildtype A375 control cells (Fig. 5d, f, g), could be blocked by prior transfection using the siPMAIP1 oligonucleotides (Fig. 5g, right panel), but was not suppressed when transfection occurred using non-targeting siRNA control reagent (siControl; Fig. 5g, middle panel).

Next, the effect of siRNA intervention targeting PMAIP1 upregulation on DHA-induced apoptosis was examined in A375 cells (Fig. 5h). Flow cytometric analysis revealed pronounced cellular protection against DHA-induced cytotoxicity (40 μM, 48 h) that was only observed in siPMAIP1-tranfected (approximately 82% survival) but not in siControl-transfected (approximately 45% survival) cells, suggesting that NOXA is a key mediator of DHA-induced apoptosis in A375 melanoma cells.

DHA impairs genomic integrity with early activational phosphorylation of p53 (p53-Ser15) in A375 melanoma cells but not in primary epidermal melanocytes

Our array-based observation that DHA treatment induced genotoxic stress response gene expression (GADD45A, CDKN1A, and DDIT3; 48 h exposure; Fig. 2) led us to examine if early induction of DNA damage may contribute to DHA-apoptogenicity in A375 metastatic melanoma cells. First, GADD45A, CDKN1A, and DDIT3 expression was examined at the transcriptional level by time course analysis using quantitative RT-PCR that revealed pronounced upregulation within 6 h (GADD45A, DDIT3) or 12 h (CDKN1A) continuous exposure (Fig. 6a–c). DHA-induced expression changes at the protein level were observed by immunoblot detection of CHOP (encoded by DDIT3/GADD153), a transcription factor that can be upregulated in response to genotoxic stress and ER stress. Using the established ER stress inducer thapsigargin (300 nM, 24 h) as a positive control, CHOP upregulation was observed within 12 continuous exposure to DHA, whereas p21 (encoded by CDKN1A) displayed increases that occurred only upon 24 h continuous exposure (Fig. 6d and e, respectively).

DHA-dependent induction of genotoxic stress was examined using alkaline single cell electrophoresis (comet assay) [23]. As evident from formation of nuclear comets in A375 cells, significant induction of genotoxic stress was detectable within 3 h exposure to low micromolar concentrations of DHA (10 μM) (Fig. 6f). At higher concentrations (40 μM), DHA treatment induced comets with average tail moments that exceeded control levels more than three fold within 6 h of exposure (Fig. 6f). In contrast, analogous comet analysis performed in human epidermal melanocytes exposed to DHA (10 and 40 μM, up to 12 h exposure time) did not reveal any impairment of genomic integrity (Fig. 6f).

DHA-induced impairment of genomic integrity was also examined by flow cytometric analysis of the nuclear phosphorylated histone variant H2A.X (γ-H2A.X, Ser 139), a sensitive marker of DNA double-strand breaks (Fig. 6g) [28]. In A375 cells, no induction of γ-H2A.X was observed in response to DHA exposure at time points and concentrations (10 μM, 3 h) that caused a significant increase in average tail moment demonstrating that DHA-induced early impairment of genomic integrity does not involve the generation of double strand breaks.

DNA damage is known to induce activational phosphorylation of tumor suppressor protein p53 at Ser15 that occurs by genotoxic stress-responsive kinases [29]. Consistent with an involvement of p53 activation that may occur upstream of GADD45A, DDIT3, and CDKN1A upregulation, early activational phosphorylation [phospho-p53 (Ser15)] was observed in response to DHA within 3 h exposure time (Fig. 6h). In contrast, treatment with DHA did not cause any changes in p53-Ser15 phosphorylation status in human epidermal melanocytes (Fig. 6h), a finding consistent with the absence of DHA-induced DNA comets observed above (Fig. 5c).